JP5030513B2 - Fluorescence observation apparatus and endoscope system for living tissue - Google Patents

Description

  The present invention relates to a fluorescence observation apparatus for a biological dye and an endoscope system.

Conventionally, in cancer, it is known that a certain type of protein is overexpressed compared to the normal part, and the cancer is diagnosed by illuminating the protein molecule expressed using a fluorescent probe. In addition, a technique for endoscopically identifying cancer by observing this fluorescence endoscopically has been proposed (see, for example, Patent Document 1 and Patent Document 2).
JP-A-6-27110 JP-A-10-201707

  However, it is difficult to diffuse the fluorescent probe with a uniform concentration distribution when the fluorescent probe is sprayed on the living tissue. In particular, since the biological tissue is not flat and has undulations, the fluorescent probe is likely to be localized. As a result, there is a problem that a fluorescence intensity distribution is generated due to the localization of the fluorescent probe, and cancer cannot be accurately identified only by the fluorescence intensity distribution.

  The present invention has been made in view of the above-described circumstances, and can detect a normal tissue and an abnormal tissue with high accuracy even when a fluorescent dye dispersed in the biological tissue is localized. And to provide an endoscope system.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to a first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a fluorescence wavelength or an absorption wavelength different from that of the first fluorescent dye. The first fluorescent dye and the second fluorescent dye are simultaneously or occasionally applied to a biological tissue to which a second fluorescent dye having a known concentration ratio and an absorption ratio to the biological tissue is attached or absorbed. An excitation light optical system that irradiates excitation light that is excited in the division; a fluorescence detection unit that detects fluorescence excited by the excitation light from the excitation light optical system; and the second that is detected by the fluorescence detection unit Provided is a fluorescence observation apparatus for biological tissue comprising a compensation processing unit that compensates for fluorescence information from the first fluorescent dye detected by the fluorescence detection unit based on information on fluorescence from the fluorescent dye.
The present invention relates to a first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a fluorescence wavelength or an absorption wavelength different from that of the first fluorescent dye. The first fluorescent dye and the second fluorescent dye are simultaneously or occasionally applied to a biological tissue to which a second fluorescent dye having a known concentration ratio and an absorption ratio to the biological tissue is attached or absorbed. Prior to detecting the fluorescence from the first fluorescent dye excited by the excitation light from the excitation light optical system and the excitation light optical system that irradiates excitation light to be excited in the division, the second A fluorescence detection unit that detects fluorescence from the fluorescent dye at least once, and the first fluorescence detected by the fluorescence detection unit based on fluorescence information from the second fluorescence pigment detected by the fluorescence detection unit Of fluorescence from fluorescent dyes Providing fluorescence observation device for living tissue and a compensation processor for compensating for.
The present invention relates to a first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a fluorescence wavelength or an absorption wavelength different from that of the first fluorescent dye. Excitation for irradiating biological tissue to which a second fluorescent dye having a known concentration ratio is attached or absorbed with excitation light that excites the first fluorescent dye and the second fluorescent dye simultaneously or in a time-sharing manner. An optical optical system, a fluorescence detection unit for detecting fluorescence excited by the excitation light from the excitation light optical system, and information on fluorescence from the second fluorescent dye detected by the fluorescence detection unit A biological tissue fluorescence observation apparatus comprising: a compensation processing unit that compensates for fluorescence information from the first fluorescent dye detected by the fluorescence detection unit.
The present invention relates to a first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a fluorescence wavelength or an absorption wavelength different from that of the first fluorescent dye. Excitation for irradiating biological tissue to which a second fluorescent dye having a known concentration ratio is attached or absorbed with excitation light that excites the first fluorescent dye and the second fluorescent dye simultaneously or in a time-sharing manner. Prior to detecting fluorescence from the first fluorescent dye excited by the excitation light from the optical optical system and the excitation light optical system, the fluorescence from the second fluorescent dye is detected at least once. And the fluorescence information from the first fluorescent dye detected by the fluorescence detection unit based on the fluorescence information from the second fluorescent dye detected by the fluorescence detection unit A compensation processing unit Providing body tissue for fluorescence observation device.

In the said invention, it is preferable that the said 1st fluorescent dye and the 2nd fluorescent dye are connected by connection means, such as a peptide chain.
In the above invention, it is preferable that the first fluorescent dye and the second fluorescent dye are excited by the same excitation light.

In the invention described above, the first fluorescent dye and the second fluorescent dye may constitute a FRET fluorescent probe.
In the above invention, the fluorescence wavelength of the first fluorescent dye may be shorter than the fluorescence wavelength of the second fluorescent dye.
Moreover, in the said invention, it is good also as the fluorescence length of a said 2nd fluorescent pigment | dye including the wavelength range | band which is 600 nm or more.

The present invention also provides an endoscope system including the above-described fluorescence observation apparatus for living tissue.
In the above invention, a discharge mechanism for discharging the first fluorescent dye and the second fluorescent dye from the tip may be provided.

Moreover, in the said invention, the said fluorescence detection part is good also as providing the etalon type | mold spectral element which separates the fluorescence from a 1st fluorescent dye and the fluorescence from a 2nd fluorescent dye.
In the above invention, the excitation unit includes the control unit and the image storage unit that stores the fluorescent image, and the control unit discharges the fluorescent dye by the discharge mechanism before the excitation light optical system and the fluorescence detection unit. The acquired autofluorescence image may be stored in the image storage unit.

In the above invention, the image forming apparatus further includes a control unit and an image storage unit that stores an image in a spectral wavelength band that reflects the blood volume in the tissue, and the control unit before discharging the fluorescent dye by the discharge mechanism. In addition, an image reflecting the blood volume may be stored in the image storage unit.
Furthermore, in the above invention, the illumination light optical system for irradiating the illumination light, and a reflected light detection unit for detecting the reflected light by the illumination light emitted from the illumination light optical system, the compensation processing unit Based on the information of the reflected light detected by the reflected light detection unit, the fluorescence information from the first fluorescent dye detected by the fluorescence detection unit may be compensated.

  According to the present invention, there is an effect that a normal tissue and an abnormal tissue can be distinguished with high accuracy even when a fluorescent dye dispersed in a living tissue is localized.

Hereinafter, a biological fluorescence observation apparatus according to a first embodiment of the present invention and an endoscope system 1 including the same will be described with reference to FIGS. 1 to 6.
The fluorescence observation apparatus for living tissue according to this embodiment is provided in the endoscope system 1 shown in FIG.

As shown in FIG. 1, an endoscope system 1 according to this embodiment includes an insertion unit 2 that is inserted into a body cavity of a living body, an imaging unit 3 that is disposed in the insertion unit 2, and a plurality of types. A light source unit 4 that emits light, a liquid supply unit (discharge mechanism) 20 that supplies liquid to be discharged from the distal end 2 a of the insertion portion 2, and a control unit 5 that controls the imaging unit 3, the light source unit 4, and the liquid supply unit 20. And a display unit 6 for displaying an image acquired by the imaging unit 3.
The biological fluorescence observation apparatus according to this embodiment includes the imaging unit 3, the light source unit 4, the control unit 5, and the display unit 6.

The insertion section 2 has a very thin outer dimension that can be inserted into a body cavity of a living body, and includes therein a light guide 7 that propagates light from the imaging unit 3 and the light source unit 4 to the tip 2a. .
The light source unit 4 illuminates the imaging target A in the body cavity and illuminates the imaging target A in the body cavity and an illumination light source 8 that emits illumination light to obtain reflected light that is reflected back from the imaging target A. And an excitation light source 9 that emits excitation light for exciting the fluorescent substance existing in the imaging target A to generate fluorescence, and a light source control circuit 10 that controls these light sources 8 and 9. .

  The illumination light source 8 is, for example, a combination of a xenon lamp and a bandpass filter (not shown), and the bandpass filter has a 50% transmission region of 410 to 430 nm, 420 to 470 nm, 500 to 580 nm, and 570 to 650 nm. is there. That is, the illumination light source 8 emits illumination light having a wavelength band of 410 to 430 nm, blue illumination light having a wavelength band of 420 to 470 nm, green illumination light having a wavelength band of 500 to 580 nm, and red illumination light having a wavelength band of 570 to 650 nm. Be able to.

  The excitation light source 9 is, for example, a semiconductor laser that emits excitation light having a peak wavelength of 490 ± 5 nm (or an argon laser that emits excitation light of 488 ± 5 nm). The excitation light having this wavelength can excite the first fluorescent dye having a fluorescein skeleton and the second fluorescent dye composed of porferin.

  As the fluorescent probe, a probe in which a first fluorescent dye and a second fluorescent dye are connected by a peptide chain, for example, is used. The second fluorescent dye made of porferin generates fluorescence having a peak in the vicinity of 650 nm when irradiated with excitation light, while the first fluorescent dye having a fluorescein skeleton does not emit fluorescence only by irradiation with excitation light. However, the first fluorescent dye generates fluorescence having a peak in the vicinity of 530 nm by irradiation with excitation light after being hydrolyzed by nonspecific esterase in an abnormal tissue such as a tumor.

The light source control circuit 10 alternately turns on and off the illumination light source 8 and the excitation light source 9 at a predetermined timing according to a timing chart described later.
As shown in FIG. 2, the imaging unit 3 includes an imaging optical system 11 that collects light incident from the imaging target A, and an excitation light cut filter 12 that blocks excitation light incident from the imaging target A. And a variable spectral element 13 whose spectral characteristics can be changed by the operation of the control unit 5 and an imaging element 14 that captures the light collected by the imaging optical system 11 and converts it into an electrical signal.

  The variable spectroscopic element 13 includes two flat plate-like optical members 13a and 13b that are arranged with a parallel interval and provided with a reflection film on the opposing surface, and an actuator 13c that changes the interval between the optical members 13a and 13b. Is an etalon type optical filter. The actuator 13c is, for example, a piezoelectric element. The variable spectroscopic element 13 can change the wavelength band of the transmitted light by changing the distance between the optical members 13a and 13b by the operation of the actuator 13c.

  More specifically, the variable spectroscopic element 13 has a transmittance wavelength characteristic having two transmission bands, one fixed transmission band and one variable transmission band, as shown in FIG. The fixed transmission band always transmits incident light regardless of the state of the variable spectroscopic element 13. Further, the transmittance characteristic of the variable transmission band changes according to the state of the variable spectroscopic element 13.

  In the present embodiment, the variable spectral element 13 has a variable transmission band in the red wavelength band (for example, 610 to 650 nm). The variable spectroscopic element 13 changes to two states according to a control signal from the control unit 5.

In the first state, the transmittance in the variable transmission band is sufficiently reduced as compared with the second state, and the passage of light in the red wavelength band is blocked.
In the second state, the transmittance of the variable transmission band is increased to 50% or more, and light in the red wavelength band is allowed to pass.

  The fixed transmission band is arranged in a range of 400 to 560 nm, and is fixed to, for example, a transmittance of 60% or more. Thereby, reflected light with respect to illumination light of 410 to 430 nm, reflected light with respect to blue and green illumination light, and fluorescence from the first fluorescent dye can be transmitted.

Thus, the variable spectroscopic element 13, when it is disposed in a first state, fixed transmission band and passing over possible, the illumination light of the wavelength band 410～430Nm, reflected light to blue and green illumination light, and the first Fluorescence from the fluorescent dye can be passed. Further, when the variable spectroscopic element 13 is arranged in the second state, in addition to the first state, the reflected light with respect to the red illumination light and the fluorescence from the second fluorescent dye can be passed. .

The excitation light cut filter 12 has a transmittance of 80% or more in the wavelength band of 400 to 460 nm, an OD value of 4 or more in the wavelength band of 480 to 500 nm (= transmittance of 1 × 10 ⁻⁴ or less), and 520 to 750 nm. It has transmittance characteristics with a transmittance of 80% or more in the wavelength band.

  As shown in FIG. 1, the control unit 5 includes an image sensor driving circuit 15 for driving and controlling the image sensor 14, a variable spectral element control circuit 16 for driving and controlling the variable spectral element 13, and a valve control circuit 25 described later. And an image storage unit 17 that stores image information acquired by the image sensor 14, and an image processing circuit 18 that processes the image information stored in the image storage unit 17 and outputs the processed image information to the display unit 6. .

  As shown in FIG. 4, the image storage unit 17 sets the input destination of the image information output from the first storage unit 17 </ b> A, the second storage unit 17 </ b> B, and the image sensor 14 to the first storage unit 17 </ b> A or the first storage unit 17 </ b> A. The first storage unit 17A includes a switch 17C that switches to the second storage unit 17B, and a plurality of frame memories 17a to 17d that store fluorescent image information and a frame memory 17e that stores image information reflecting blood volume. The second storage unit 17B includes a plurality of frame memories 17f to 17h that store white observation image information.

In the frame memory 17a of the first storage unit 17A, autofluorescence image information Ga obtained by irradiating excitation light before the fluorescent probe is dispersed and the variable spectroscopic element 13 in the first state is stored. ing.
The autofluorescence image information Ga acquired in a state where no drug fluorescence is generated before the fluorescent probe is dispersed is acquired in the first state in which the drug fluorescence from only the first fluorescent dye is transmitted. It becomes a background image of a fluorescent image containing drug fluorescence from only the fluorescent dye.

The frame memory 17b stores autofluorescence image information Gb obtained by irradiating excitation light before the fluorescent probe is dispersed and acquiring the variable spectroscopic element 13 in the second state.
Since the autofluorescence image information Gb is acquired in the second state in which all of the drug fluorescence from the first and second fluorescent dyes is transmitted, the background image of the fluorescence image including the drug fluorescence from both fluorescent dyes It becomes.

  The frame memory 17c stores fluorescence image information Gc obtained by irradiating excitation light after spraying fluorescent probes and acquiring the variable spectral element 13 in the first state. The fluorescence image information Gc is fluorescence image information obtained by photographing the drug fluorescence and autofluorescence from only the first fluorescent dye.

  The frame memory 17d stores fluorescence image information Gd obtained by irradiating excitation light after spraying fluorescent probes and acquiring the variable spectroscopic element 13 in the second state. This fluorescence image information Gd is fluorescence image information obtained by photographing the drug fluorescence and autofluorescence from the first and second fluorescent dyes.

The frame memory 17e stores a reflected light image Ge acquired by setting the variable spectral element 13 in the second state and irradiating illumination light with a wavelength of 410 to 430 nm.
Since the wavelength band 410 to 430 nm of the illumination light includes the absorption band of hemoglobin, imaging the reflected light reflects the blood volume such as the structure of blood vessels that are relatively close to the surface of the living tissue. Image information can be acquired.

  Further, in the frame memories 17f to 17h of the second storage unit 17B, as shown in FIG. 5, the variable spectroscopic element 13 is set to the second state, and the blue light and the green light are reflected in the reflected light image for white observation. Reflected light image information obtained by irradiating light and red light, respectively, is stored.

The imaging element driving circuit 15 and the variable spectral element control circuit 16 are connected to the light source control circuit 10 and are synchronized with the switching of the illumination light source 8 and the excitation light source 9 by the light source control circuit 10. The image pickup device 14 is driven and controlled.
Specifically, as shown in the timing chart of FIG. 6, when the excitation light source or the illumination light having the wavelength band of 410 to 430 nm is emitted from the excitation light source 9 by the operation of the light source control circuit 10, the variable spectroscopic element control is performed. The circuit 16 switches the variable spectroscopic element 13 between the first state and the second state, the switch 17C is switched to the first storage unit 17A side, and the image sensor driving circuit 15 is output from the image sensor 14. Image information is output to the first storage unit 17A.

  When the illumination light source 8 emits illumination light for white observation by the operation of the light source control circuit 10, the variable spectroscopic element control circuit 16 sets the variable spectroscopic element 13 to the second state and switches 17C. Is switched to the second storage unit 17B side, and the image sensor drive circuit 15 outputs the image information output from the image sensor 14 to the second storage unit 17B.

In addition, in the case of white observation, the image processing circuit 18 receives reflected light image information obtained by irradiation of blue, green, and red illumination light from the frame memories 17f to 17h of the second storage unit 17B. The data is output to the three channels R, G, and B of the display unit 6.
In the case of fluorescence observation, the image processing circuit 18 receives fluorescence image information obtained by irradiation with excitation light from the first storage unit 17A, and performs the following inter-image calculation processing.

G ₁ = Gc-Ga
G ₂ = Gd−Gb
G ₃ = G ₂ −G ₁
G ₄ = G ₁ / G ₃

Calculated fluorescence image information G ₁ is obtained by removing the autofluorescence image Ga is its background from the fluorescent image Gc of the first fluorescent dye shows fluorescence intensity of only the first fluorescent dye.
Fluorescence image information G ₂ is is obtained by removing the autofluorescence image Gb its background from the fluorescent image Gd of the first and second fluorescent dyes, shows a fluorescence intensity of the first and second fluorescent dye Yes.

Fluorescence image information G ₃ are, which from the fluorescence image information G ₂ to remove the fluorescence image information G _1, shows the fluorescence intensity of only the second fluorescence dye.
Fluorescence image information G ₄ are is obtained by dividing the fluorescence image information G ₁ in the fluorescence image information G _3, the fluorescence intensity of the first fluorescence dye, has been normalized by the fluorescence intensity of the second fluorescence dye.
Then, for example, the image processing circuit 18 outputs the fluorescence image information G ₁ , G ₃ , and G ₄ obtained by the irradiation of excitation light to the three channels R, G, and B of the display unit 6, respectively. It has become.

In the present embodiment, the second fluorescence wavelength band exists on the long wavelength side that is not easily affected by blood absorption. However, the fluorescence wavelength band of the first excitation light exists in a region that is strongly absorbed by blood. Generally, blood is increased in tumors and cancers compared to normal tissues, and quantitativeness is lost unless this absorption is taken into account.
Therefore, in this embodiment, by using a Ge image is a reflection light image which is capable of reflecting particularly strong blood flow in the mucosal surface 410Nm～430nm, previously normalized fluorescence image information G ₁ described above.
That is,
_{G 1 = (Gc-Ga)} / Ge
If so, the influence of absorption by blood can be corrected.

  The liquid feeding unit 20 is connected to the tank 21 for storing the fluorescent probe liquid, the valve 22 for supplying / stopping the liquid from the tank 21, and the valve 22 to the tip 2a along the insertion portion 2. A liquid feeding tube 23 to be supplied and a valve control circuit 25 arranged in the control unit 5 and controlling the valve 22 are provided. The liquid delivery tube 23 has its distal end disposed at the distal end 2 a of the insertion portion 2, and the sent fluorescent probe can be dispersed toward the imaging target A. As the liquid feeding tube 23, a forceps channel provided in the insertion portion 2 may be used.

As shown in FIG. 6, the bulb control circuit 25 is a fluorescent probe solution stored in the tank 21 for a predetermined time during white light observation before activation of the excitation light source 9 for obtaining a fluorescence image of a drug. The valve 22 is controlled so as to be sprayed.
In addition, the valve control circuit 25 is configured to switch the valve 22 to an off state after spraying the fluorescent probe liquid.

The operation of the endoscope system 1 according to the present embodiment configured as described above will be described below.
In order to image the imaging target A in the body cavity of the living body using the endoscope system 1 according to the present embodiment, first, the insertion unit 2 is inserted into the body cavity, and the distal end 2a thereof is the imaging target A in the body cavity. To face. In this state, the light source unit 4 and the control unit 5 are operated, and the light source control circuit 10 is operated to operate the excitation light source 9 to generate excitation light.

At this time, the variable spectroscopic element 13 is first set to the first state by the operation of the variable spectroscopic element control circuit 16 of the control unit 5, and then switched to the second state after a predetermined time has elapsed.
Excitation light generated in the light source unit 4 is propagated to the distal end 2a of the insertion portion 2 via the light guide 7, and is irradiated toward the imaging target A from the distal end 2a of the insertion portion 2.
When the imaging object A is irradiated with the excitation light, the autofluorescent substance that is naturally present in the imaging object A is excited and emits autofluorescence. The autofluorescence emitted from the imaging target A is collected by the imaging optical system 11 of the imaging unit 3, passes through the excitation light cut filter 12, and enters the variable spectral element 13.

  During the period when the variable spectral element 13 is switched to the first state, only light in the fixed transmission band can be transmitted. Therefore, only autofluorescence in the fixed wavelength band is transmitted through the variable spectral element 13 and imaged. Taken by unit 3. The fluorescence image information Ga acquired by the image sensor 14 is stored in the frame memory 17a of the first storage unit 17A. In this case, a part of the excitation light irradiated to the imaging target A is reflected by the imaging target A and enters the imaging unit 3 together with the fluorescence. The imaging unit 3 is provided with the excitation light cut filter 12. Therefore, the excitation light is blocked and is prevented from entering the image sensor 14.

  On the other hand, when the variable spectroscopic element 13 is switched to the second state, not only the fixed transmission band but also the light in the variable transmission band can be transmitted. The image is taken by the imaging unit 3 through the element 13. Then, the fluorescence image information Gb acquired by the image sensor 14 is stored in the frame memory 17b of the first storage unit 17A.

  Thereafter, the excitation light source 9 is stopped by the light source control circuit 10 and the illumination light source 8 is activated to emit illumination light for white observation, and the bulb 21 is opened by the operation of the bulb control circuit 25. A fluorescent probe solution is sprayed. At this time, the reflected light image information acquired by the imaging unit 3 is input to the frame memories 17f to 17h of the second storage unit 17B, and is output and displayed on the three channels R, G, and B of the display unit 6. The

  Thereafter, the light source control circuit 10 switches the illumination light source 8 to the excitation light source 9 again. First, the variable spectroscopic element control circuit 16 switches the variable spectroscopic element 13 to the first state. The fluorescent image information Gc by the fluorescent dye is acquired, and then the variable spectral element is switched to the second state to acquire the fluorescent image information Gd by the first and second fluorescent dyes.

Then, the image processing circuit 18 receives the fluorescence image information Ga~Gd obtained by irradiation of the excitation light from the first storage section 17A, performs image processing, the fluorescence image information _{G 1,} G 3, _{G 4} Calculate and output to the R, G and B channels of the display unit 6, respectively. Thereby, the fluorescence image information G ₁ , G ₃ , G ₄ is displayed on the display unit 6, respectively.

In this case, according to the present embodiment, the fluorescence image information G1 is calculated by removing the autofluorescence image information Ga that has passed through the fixed transmission band from the fluorescence image information Gc from the first fluorescent dye. . Therefore, it is possible to observe the fluorescence image information G1 using only the first fluorescent dye that is not affected by autofluorescence.

According to the present embodiment, the fluorescence image information G3 removes the autofluorescence image information Gb that has passed through the fixed transmission band and the variable transmission band from the fluorescence image information Gd from the first and second fluorescent dyes, Furthermore, it is calculated by removing the fluorescence image G1 with only the first fluorescent dye. Therefore, it is possible to observe the fluorescence image information G3 using only the second fluorescent dye that is not affected by autofluorescence.

Furthermore, according to the present embodiment, a fluorescent probe in which a first fluorescent dye having a fluorescein skeleton and a second fluorescent dye made of porferin are combined at a concentration ratio of 1: 1 by a peptide chain is used. since it is, and can be a fluorescence image information G ₁ from the first fluorescence dye, to obtain a fluorescent image information G ₄ which is compensated by using the fluorescence image information G ₃ from the second fluorescence dye.

  That is, the first fluorescent dye undergoes a hydrolysis reaction by a nonspecific esterase in an abnormal tissue in a living tissue, for example, a tumor part, and emits strong fluorescence when irradiated with excitation light. On the other hand, the second fluorescent dye emits fluorescence when irradiated with excitation light regardless of the tissue in the living tissue.

Accordingly, the fluorescence image information G ₃ from the second fluorescence dye, becomes information indicating the localization of the second fluorescent dye, a concentration ratio of 1: the localization of the first fluorescent dye coupled with 1 It becomes information to show. As a result, the fluorescence image information G ₁ from the first fluorescence dye, by dividing the fluorescence image information G ₃ from the second fluorescence dye, the fluorescence image information G ₄ which has been compensated for localization of fluorescent dyes Obtainable.
That is, according to the biological fluorescence observation apparatus and the endoscope system 1 according to the present embodiment, it is possible to accurately distinguish between a normal tissue and an abnormal tissue even if the fluorescent pigment dispersed in the biological tissue is localized. There is an advantage that you can.

In the present embodiment, the excitation light and the reflected light are sequentially switched by the operation of the light source control circuit 10 to perform the fluorescence observation and the white observation. In the fluorescence observation, the fluorescence image information Ga˜ Based on Gd, the corrected fluorescence image information G ₁ , G ₃ , G ₄ is calculated. Alternatively, for fluorescence image information G ₃ for compensating the localization, the fluorescence image information Ga~Gd acquired by radiating excitation light immediately after spraying fluorescent probes or fluorescent image information Ga～, stores the fluorescence image information G ₃ calculated using gd, the stored fluorescence image information Ga-gd, the G ₃ may be used for calculating the subsequent fluorescence image information G _4.
By doing so, even if the fluorescence from the second fluorescent dye changes due to discoloration or wavelength change, the localization of the first fluorescent dye can be compensated and the abnormal tissue can be detected with high accuracy. There is an advantage.

  In the present embodiment, a fluorescent probe in which a first fluorescent dye having a fluorescein skeleton and a second fluorescent dye made of porferin are connected by a peptide chain is used. Alternatively, a fluorescent probe in which any two fluorescent dyes having different absorption wavelengths and known concentration ratios and absorption ratios to living tissues may be used.

In the present embodiment, the fluorescence image information G ₁ , G ₃ , G ₄ calculated as described above is displayed. However, when it is desired to observe the acquired image in real time, an inter-image calculation process is performed. The fluorescent image information Gc, Gd, and Ge may be simply output to the R, G, and B channels of the display unit 6 without performing the above.

Next, a biological fluorescence observation apparatus and an endoscope system according to a second embodiment of the present invention will be described below with reference to FIGS.
In the description of the biological fluorescence observation apparatus and the endoscope system according to the present embodiment, the parts having the same configurations as those of the biological fluorescence observation apparatus and the endoscope system 1 according to the first embodiment described above are provided. The same reference numerals are given and the description is omitted.

The endoscope system according to the present embodiment is different in the fluorescent probe to be used, and accordingly, the excitation light source 9 and the transmittance characteristics of each member of the imaging unit 3 and the control method by the control unit 5 are different. ing.
In the present embodiment, a FRET fluorescent probe having Cy5.5 (Amersham) as a donor and Cy7 (Amersham) as an acceptor is used as a fluorescent probe.

  When this fluorescent probe is irradiated with excitation light of 660 nm immediately after being spread on a living tissue, Cy5.5 (first fluorescent dye) as a donor does not emit fluorescence due to fluorescence resonance energy transfer (FRET), and Cy7 Only (second fluorescent dye) emits fluorescence. Then, when the connection between the two fluorescent dyes is cut by the action of the target enzyme (for example, esterase etc.), the fluorescence resonance energy does not move, the fluorescence of the acceptor Cy7 disappears, and the donor Cy5. 5 starts to emit fluorescence.

The excitation light source 9 is, for example, a semiconductor laser that emits excitation light having a peak wavelength of 660 ± 5 nm. The excitation light of this wavelength can excite the fluorescent probe containing the first fluorescent dye and the second fluorescent dye.
As shown in FIG. 7, the variable spectroscopic element 13 has a transmittance wavelength characteristic having two transmission bands, one fixed transmission band and one variable transmission band. The fixed transmission band always transmits incident light regardless of the state of the variable spectroscopic element 13. Further, the transmission band of the variable transmission band changes according to the state of the variable spectroscopic element 13.

In the present embodiment, the variable spectroscopic element 13 is configured to move the variable transmission band into two states according to a control signal from the control unit 5.
That is, the first state is a state in which the variable transmission band matches the wavelength band (for example, 685 to 715 nm) of fluorescence from the first fluorescent dye. Thereby, the fluorescence from the first fluorescent dye can be transmitted.
The second state is a state in which the variable transmission band coincides with the wavelength band (for example, 720 to 760 nm) of the fluorescence from the second fluorescent dye. Thereby, the fluorescence from the second fluorescent dye can be transmitted.

The fixed transmission band is arranged in the range of 400 to 650 nm, for example, and is fixed at a transmittance of 60% or more.
Further, the fixed transmission band is located in a wavelength band including the wavelength of the self-fluorescence on the short wavelength side and the wavelength of the reflected light with respect to the illumination light. In either case of the first and second states, the self-transmission on the short wavelength side Fluorescence and reflected light can be transmitted toward the image sensor 14.

The excitation light cut filter 12 has a transmittance of 80% or more in the wavelength band of 400 to 640 nm, an OD value of 4 or more in the wavelength band of 650 to 670 nm (= transmittance of 1 × 10 ⁻⁴ or less), and 680 to 750 nm. It has transmittance characteristics with a transmittance of 80% or more in the wavelength band.

The operation of the endoscope system according to this embodiment configured as described above will be described below.
In order to image the imaging target A in the body cavity of the living body using the endoscope system according to the present embodiment, first, the insertion portion 2 is inserted into the body cavity, and the distal end 2a is set as the imaging target A in the body cavity. Make them face each other. In this state, the light source unit 4 and the control unit 5 are operated, and the light source control circuit 10 is operated to operate the excitation light source 9 to generate excitation light.

  At this time, the variable spectroscopic element 13 is first set to the first state by the operation of the variable spectroscopic element control circuit 16 of the control unit 5, and then switched to the second state after a predetermined time has elapsed. As a result, the two types of autofluorescence image information Ga and Gb for autofluorescence compensation are acquired in the same manner as in the first embodiment.

In this embodiment, the excitation light source 9 is then stopped by the light source control circuit 10 and the illumination light source 8 is activated to irradiate illumination light in the wavelength band 410 to 430 nm, and the reflected light image reflecting the blood volume. Information Ge is acquired and stored in the frame memory 17e.
Further, after that, the light source control circuit 10 activates the illumination light source 8 to emit illumination light for white observation, and the bulb 21 is opened by the operation of the bulb control circuit 25, and the fluorescent probe liquid is sprayed. At this time, the reflected light image information acquired by the imaging unit 3 is input to the frame memories 17f to 17h of the second storage unit 17B, and is output and displayed on the three channels R, G, and B of the display unit 6. The

In the present embodiment, immediately after the fluorescent probe is sprayed on the living tissue, the light source control circuit 10 is switched again from the illumination light source 8 to the excitation light source 9, and the variable spectroscopic element 13 is changed to the first one. The fluorescent image information Gd by the second fluorescent dye is acquired in the state of 2. Next, the variable spectroscopic element control circuit 16 switches the variable spectroscopic element 13 to the first state, and the fluorescence image information Gc by the first fluorescent dye is acquired.

Thereafter, the image processing circuit 18 performs image processing on the obtained fluorescence image information Ga to Gd, calculates fluorescence image information G ₁ , G ₃ , and G _4, and outputs them to the R, G, and B channels of the display unit 6, respectively. As a result, the fluorescence image information G ₁ , G ₃ , and G ₄ are displayed on the display unit 6 in the same manner as in the first embodiment.

According to the present embodiment, fluorescent image information Ga and Gb for autofluorescence compensation is acquired before the fluorescent probe is distributed, and fluorescent image information Gd by the second fluorescent dye is acquired immediately after the fluorescent probe is distributed. Thereafter, only the acquisition of the fluorescence image information Gc by the first fluorescent dye is sequentially performed. Therefore, since the fluorescence image information G ₁ , G ₃ , G ₄ is calculated by updating only the fluorescence image information Gc, it can be updated quickly.

Further, according to the present embodiment, the fluorescence image information G ₃ and G ₄ are calculated using the fluorescence image information Gd acquired immediately after the fluorescent probe is dispersed and stored in the frame memory 17d. Therefore, even if the fluorescence from the second fluorescent dye is discolored or changes in wavelength, the localization of the fluorescent dye can be compensated without being affected by this, and the abnormal tissue can be accurately identified. There is an advantage.

  In the present embodiment, the fluorescence image information Gd is acquired only once immediately after the fluorescent probe is dispersed. Alternatively, the fluorescence image information Gd may be acquired twice or more. Further, when it is considered that the influence of fading or the like is small, the fluorescence image information Gd may be obtained every time the fluorescence image information Gc is obtained, as indicated by a chain line in FIG.

In this embodiment, an etalon (Fabry-Perot) type variable spectroscopic element is used as the variable spectroscopic element. However, instead of this, another variable spectral filter may be used, and the spectral characteristic may be fixed.
Further, the fluorescence endoscope system of the present invention is not limited to a scope type having the imaging means 14 at the distal end of the insertion portion 2 to be inserted into a body cavity of a living body, but a light source portion, imaging means, and variable spectral means. May be provided in a single casing, and the casing may be inserted into a body cavity of a living body.
Furthermore, the fluorescence observation apparatus for living body of the present invention may be applied not only to a device inserted into a living body but also to an apparatus that performs fluorescence observation outside the living body.

  Fluorescein includes lactone-type fluorescein and quinoid-type fluorescein. It is known that the absorption band of excitation light can be changed by hydrolysis from lactone-type to quinoid-type fluorescein. Therefore, if lactone type fluorescein is used as the first fluorescent dye and quinoid type fluorescein is used as the second fluorescent dye, the absorption wavelength (excitation light) of each fluorescent dye can be changed without changing the fluorescence detection wavelength band. It is easy to imagine that the same thing can be done by changing the wavelength band.

1 is a block diagram showing an overall configuration of an endoscope system including a biological fluorescence observation apparatus according to a first embodiment of the present invention. It is a schematic block diagram which shows the structure inside the imaging unit of the endoscope system of FIG. It is a figure which shows the transmittance | permeability characteristic of each optical component which comprises the endoscope system of FIG. 1, and the wavelength characteristic of irradiation light and fluorescence. It is a block diagram which shows the internal structure of the image memory | storage part of the endoscope system of FIG. It is a timing chart explaining the operation | movement at the time of white light observation of the endoscope system of FIG. It is a timing chart explaining the operation | movement at the time of fluorescence observation of the endoscope system of FIG. It is a block diagram which shows the whole structure of an endoscope system provided with the fluorescence observation apparatus for biological bodies which concerns on the 2nd Embodiment of this invention. It is a timing chart explaining the operation | movement at the time of fluorescence observation of the endoscope system of FIG.

Explanation of symbols

Ga, Gb autofluorescence image information (autofluorescence image)
Ge reflected light image (blood volume reflection image)
1 Endoscope system 3 Imaging unit (fluorescence detector)
7 Light guide (Excitation light optical system)
9 Light source for excitation light (excitation light optical system)
13 Variable spectral element (spectral element)
17 Image storage unit 18 Image processing unit (compensation processing unit)
20 Liquid feed unit (discharge mechanism)

Claims (15)

A first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a fluorescence wavelength or an absorption wavelength different from each other and a concentration ratio to the first fluorescent dye; The first fluorescent dye and the second fluorescent dye are excited simultaneously or in a time-division manner with respect to the biological tissue to which the second fluorescent dye having a known absorption ratio to the biological tissue is attached or absorbed. An excitation light optical system for irradiating the excitation light;
A fluorescence detection section that detecting the excited fluorescence by the excitation light from the excitation light optical system,
A compensation processing unit that compensates for fluorescence information from the first fluorescent dye detected by the fluorescence detection unit based on fluorescence information from the second fluorescent dye detected by the fluorescence detection unit; A fluorescence observation apparatus for living tissue. A first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a fluorescence wavelength or an absorption wavelength different from each other and a concentration ratio to the first fluorescent dye; The first fluorescent dye and the second fluorescent dye are excited simultaneously or in a time-division manner with respect to the biological tissue to which the second fluorescent dye having a known absorption ratio to the biological tissue is attached or absorbed. An excitation light optical system for irradiating the excitation light;
Prior to detecting fluorescence from the first fluorescent dye excited by the excitation light from the excitation light optical system, a fluorescence detection unit for detecting fluorescence from the second fluorescent dye at least once; ,
A compensation processing unit that compensates for fluorescence information from the first fluorescent dye detected by the fluorescence detection unit based on fluorescence information from the second fluorescent dye detected by the fluorescence detection unit; A fluorescence observation apparatus for living tissue. A first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a different fluorescence wavelength or absorption wavelength and a known concentration ratio with respect to the first fluorescent dye An excitation light optical system that irradiates the biological tissue to which the second fluorescent dye is attached or absorbed with excitation light that excites the first fluorescent dye and the second fluorescent dye simultaneously or in a time-division manner; ,
A fluorescence detection section that detecting the excited fluorescence by the excitation light from the excitation light optical system,
A compensation processing unit that compensates for fluorescence information from the first fluorescent dye detected by the fluorescence detection unit based on fluorescence information from the second fluorescent dye detected by the fluorescence detection unit; A fluorescence observation apparatus for living tissue. A first fluorescent dye that selectively stains normal tissue and abnormal tissue in a living tissue, and the first fluorescent dye has a different fluorescence wavelength or absorption wavelength and a known concentration ratio with respect to the first fluorescent dye An excitation light optical system that irradiates the biological tissue to which the second fluorescent dye is attached or absorbed with excitation light that excites the first fluorescent dye and the second fluorescent dye simultaneously or in a time-division manner; ,
Prior to detecting fluorescence from the first fluorescent dye excited by the excitation light from the excitation light optical system, a fluorescence detection unit for detecting fluorescence from the second fluorescent dye at least once; ,
A compensation processing unit that compensates for fluorescence information from the first fluorescent dye detected by the fluorescence detection unit based on fluorescence information from the second fluorescent dye detected by the fluorescence detection unit; A fluorescence observation apparatus for living tissue. The fluorescence observation apparatus for living tissue according to claim 1 or 3, wherein the first fluorescent dye and the second fluorescent dye are connected by a peptide chain connecting means.   The fluorescence observation apparatus for living tissue according to claim 2 or 4, wherein the first fluorescent dye and the second fluorescent dye constitute a FRET type fluorescent probe.   The fluorescence observation apparatus for living tissue according to any one of claims 1 to 4, wherein the first fluorescent dye and the second fluorescent dye are excited by the same excitation light.   The fluorescence observation apparatus for living tissue according to any one of claims 1 to 4, wherein a fluorescence wavelength of the first fluorescent dye is shorter than a fluorescence wavelength of the second fluorescent dye.   The fluorescence observation apparatus for biological tissue according to claim 8, wherein the fluorescence wavelength of the second fluorescent dye includes a wavelength band of 600 nm or more.   An endoscope system comprising the fluorescence observation device for living tissue according to any one of claims 1 to 4.   The endoscope system according to claim 10, further comprising a discharge mechanism that discharges the first fluorescent dye and the second fluorescent dye from a tip.   The endoscope system according to claim 10, wherein the fluorescence detection unit includes an etalon-type spectroscopic element that separates fluorescence from the first fluorescent dye and fluorescence from the second fluorescent dye. A control unit and an image storage unit for storing a fluorescent image;
The control unit operates the excitation light optical system and the fluorescence detection unit before discharging the fluorescent dye by the discharge mechanism, and stores the acquired autofluorescence image in the image storage unit. Endoscope system. A control unit, and an image storage unit that stores an image in a spectral wavelength band that reflects the blood volume in the tissue,
The endoscope system according to claim 10, wherein the controller stores an image reflecting the blood volume in the image storage unit before discharging the fluorescent dye by the discharge mechanism. An illumination light optical system that irradiates illumination light, and a reflected light detection unit that detects reflected light from the illumination light emitted from the illumination light optical system,
5. The compensation processing unit compensates fluorescence information from the first fluorescent dye detected by the fluorescence detection unit based on information on reflected light detected by the reflected light detection unit. The fluorescence observation device for living tissue according to any one of the above.

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