(19) Japan
`Patent office
`(JP)
`
`(12) Japanese Laid-Open Patent (A)
`.
`
`(11) Japan Patent
`Laid-Open Number
`
`No. H9-309845
`
`(43) Date of publication of application: December 2, 1997
`
`(51)
`Lnt.C
`l.
`
`«5
`
`A61K 49/00
`
`A61B 10/00
`
`Identification
`Symbol
`
`Internal
`File No.
`
`FI
`
`A61K 49/00
`
`A613
`
`10/00
`
`A
`
`E
`
`Technical
`display
`place
`Field
`
`Request for Examination: Unrequested
`Number of Claims; 5 FD (13 pages in all)
`
`(21) Application. No.
`
`H8-149959
`
`(22) Date of filing;
`
`May 21, 1996
`
`(71)
`
`.Applicant; 000236436
`Hamamatsu Photonics K.K.
`1126-1, Ichinocho, Kamamatsu-shi,
`Shizuoka-ken
`(72)
`Inventor; Masataka Jibu
`c/o Hamamatsu Photonics K.K,
`1126-1, Ichinocho, Hamamatsu-shi,
`Shizuoka-ken
`(72) Inventor; Kaoru Sakatani
`YinHua DongLu, HepingLi, Beijing,
`The People's Republic of China 100029
`(74) Agent; Patent Attorney,
`Yoshiki Hasegawa (and another four)
`
`(54) [Title of the Invention,]
`
`NEAR- INFRARED RAY FLUORESCENT TRACER AND FLUORESCENCE IMAGING METHOD
`
`(57) [Abstract]
`
`[Problem to be Solved]
`
`The invention provides a near-infrared ray fluorescent tracer and fluorescence imaging method.
`
`[Solution]
`
`A near-infrared ray fluorescent tracer involving the present invention comprises a complex of at least a near-
`
`infrared ray fluorescent pigment and a substance that contains fat-soluble ingredients, furthermore comprises a detection
`
`target identifying part. Furthermore the near-infrared ray fluorescent tracer is such that the near-infrared ray
`
`fluorescent pigment is indocyanine green pigments and the substance that contains a fat-soluble ingredient is a high
`
`density lipoprotein. Moreover, the near-infrared ray fluorescent tracer is such that the detection target identifying
`
`part is antibodies.
`
`In an ex vivo fluorescence imaging method of the present invention, the near-infrared ray fluorescent tracer is
`
`introduced into a living body, the living body is irradiated with excitation light, and the near-infrared ray fluorescent
`
`light is detected from the tracer.
`
`Note: For Figure 1, see the back of the document
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`[Claims for the Patent]
`
`[Claim l]
`
`A near-infrared ray fluorescent tracer comprising a complex of at least near-infrared ray fluorescent
`
`pigments and a substance that contains a fat--soluble ingredient.
`
`[Claim 2]
`
`A near-infrared ray fluorescent tracer comprising at least a near- infrared ray fluorescent pigment, a
`
`substance that contains a fat-soluble ingredient, and a detection target identifying part.
`
`[Claim 3]
`
`The near-infrared ray fluorescent tracer according to claim 1 or 2, wherein
`
`the near-infrared ray fluorescent pigment is indocyanine green pigment and the substance that contains a fat-
`
`soluble ingredient is a high density lipoprotein,
`
`[Claim 4]
`
`The near-infrared ray fluorescent tracer according to claim 2 or 3, wherein
`
`the detection target identifying part is antibodies.
`
`[Claim 5]
`
`An ex vivo fluorescence imaging method comprising:
`
`introducing the near-infrared ray fluorescent tracer of any of claims 1 to 4 into a living body;
`
`irradiating the living body with excitation light; and
`
`detecting near-infrared ray fluorescent light from the near-infrared ray fluorescent tracer.
`
`[Detailed Description of the Invention]
`
`[0001]
`
`[Field of the Invention]
`
`The present invention relates to a near-infrared ray fluorescent tracer.
`
`[0002]
`
`[Prior Art]
`
`Methods in which ex vivo measurements of an internal part of thick living sample (a human body for instance)
`
`performed using light are important in medical research and in diagnosis and treatment of diseases. Knowing in advance
`
`the position and size of the tumors in particular using image diagnosis is an important technique for extracting tumors,
`
`and several methods are well-known.
`
`[0003]
`
`However, the image diagnosis of nerve axonal flow and tumor tissue using radioactive isotopes (RI) has problems such as
`
`worries about exposure and contamination, and the problem is the management is complex. Also, since this cannot be used
`
`outside controlled areas, the application in an operating time is difficult, posing problems. For instance, substance is
`
`administered in proximity of the neuromuscular junction, a phenomenon is known that the substance is absorbed by nerve cells
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`which actively transport the substance from the nerve endings towards the cell body (nerve axonal flow, if the speed of the
`
`nerve axonal flow is slower than normal, it is known that it is possible to diagnose that the nerve cells have been damaged
`
`in some way. There have been actual attempts to perform this type of diagnosis using an RI as the substance, but it has the
`
`similar problems as tumor diagnosis.
`
`[0004] Moreover, Regarding X ray CT techniques, the issue of being exposed to radiation is the same as the case of
`
`radioisotopes. Another problem is that, since the devices used are large and a test subject must be positioned deep within a
`
`tomography device, there also exists issues in that it is difficult to apply these techniques during surgery,
`
`[0005]
`
`[Problems to be Solved by the Invention]
`
`The present invention has been executed in view of the above-described problems. Specifically, it provides a
`
`novel tracer having a low toxicity which can be used against the living body for extracorporeally measuring the inner
`
`state of a living body sample having thickness (such as human body) with near-infrared ray fluorescence. It also
`
`provides an extracorporeal measuring and imaging method using the tracer.
`
`[0006]
`
`[Means for Solving the Objective Problem]
`
`It is thought that since near-infrared ray light is highly permeable to the living body, a pigment emitting
`
`the near-infrared ray light is allowed to be distributed within the body and its extracorporeal measurement can be
`
`carried out for application to a variety of diagnoses and others. Further, this method can be accomplished by the
`
`configuration of only small and inexpensive devices such as a halogen lamp, CCD camera, optical filter and a lens
`
`etc.
`
`[0007]
`
`On the other hand, the pigment poses a problem of toxicity when it is used as a tracer in the living body, and
`
`the pigments that can be used are limited. In view of this toxicity problem, indocyanine green has actually been put
`
`to use in clinical application as one that can be used as a tracer pigment.
`
`The pigment discharges near red fluorecent light (835nm) in organic solvent, and there is an instance in which
`
`using this, blood vessels at the bottom of the eye was observed fluorecently.
`
`[0008]
`
`However, though the pigment is fluorescent in a non-polar solvent (such as an organic solvent), the pigment is
`
`non-fluorescent in a polar solvent (such as an aqueous solution), and in an unaltered form the pigment described above
`
`cannot be used as is.
`
`[0009]
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`The inventor diligently researched the above-described points, and succeeded in discovering a new near-infrared ray
`
`fluorescent tracer that contains a non-toxic near-infrared ray fluorescent pigment and is water soluble.
`
`That is, the inventor discovered that when a pigment, such as indocyanine green pigment, that is low in toxicity but
`
`substantially non- fluorescent in aqueous solution, forms a complex with an appropriate high-density lipoprotein or the
`
`like, it was fluorescent. Based on this discovery, the inventor succeeded in making the complex described above as a near-
`
`infrared ray fluorescent tracer. The inventor also discovered a near-infrared ray fluorescent pigment tracer further with
`
`an identifying part capable of uniquely identifying a detection target. Based on this discovery, the inventor succeeded in
`
`making the complex into a near-infrared ray fluorescent tracer capable of uniquely identifying the detection target.
`
`[0010]
`
`More specifically, the present invention provides a near-infrared ray fluorescent tracer comprising a complex
`
`that contains at least a near- infrared fluorescent pigment and R substance that contains a fat-soluble ingredient.
`
`[0011]
`
`The present invention also provides a near-infrared ray fluorescent tracer comprising at least a near-infrared
`
`ray fluorescent pigment, a substance that contains a fat-soluble ingredient, and a detection target identifying part.
`
`[0012]
`
`Furthermore, the present invention provides a near-infrared ray fluorescent tracer wherein the near-infrared ray
`
`fluorescent pigment is an indocyanine green pigment and the substance that contains a fat-soluble ingredient is a
`
`high-density lipoprotein.
`
`[0013]
`
`The present invention further provides a near-infrared ray fluorescent tracer wherein the detection target
`
`identifying part is an antibody.
`
`[0014]
`
`Furthermore, this invention provides an extracorporeal fluorescent imaging method comprising introducing a near-
`
`infrared ray fluorescent tracer into a living body, irradiating the living body with an excitation light and detecting
`
`near-infrared ray fluorescence from the tracer.
`
`[0015]
`
`The following embodiments are provided for a more detailed description of the present invention.
`
`[0016]
`
`[Embodiments of the Invention]
`
`(Near-infrared ray tracer invention) The near-infrared ray tracer of the present invention contains a pigment that has
`
`fluorescent wavelengths in the near-infrared ray band, and that is traceable through a detection of the fluorescence. Here,
`
`the near-infrared ray band should be at least 700nm, more preferably 800 nm or more of the range, and the upper limit is not
`
`specified, but in the actual organic pigment, it is in the range of 1200 nm ~ 1600 nm. As to the pigment itself, it is
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`necessary that it has fluorescence in the medium where the target detection object exists, but as explained in the following,
`
`it is not always necessary to be fluorescent in water soluble liquid.
`
`[0017]
`
`Moreover, Since the tracer of the present invention is to be used inside a living body, particularly required
`
`characteristics for the tracer include solubility in water (so as to dissolve in vivo liquid medium), and being non-
`
`toxic to the living body. As long as the pigment combines the characteristics described above, in the present invention,
`
`there are no other restrictions on the pigment. A pigment such as indocyanine green pigment can be used favorably.
`
`Indocyanine green pigment is water soluble. Also, indocyanine green is used as liver circulatory function detection test
`
`drug, and so toxicity does not present a problem.
`
`[0018]
`
`The near-infrared ray tracer according to this invention is preferably a complex with the pigment bonded to other
`
`living ingredient. Among the other living ingedients which can be bonded by the pigment, there are no particular
`
`restrictions and various biological components can be selected depending on the application of the tracer according to
`
`the invention. For example, (i) when the pigment described above is insoluble in an in vivo liquid medium (e.g., blood
`
`or spinal fluid), complexes with different living substances can be formed to make the pigment soluble; (ii) conversely,
`
`in spite of the fact that
`
`the pigment described above is soluble in the in vivo liquid medium (e.g., blood or spinal
`
`fluid), but in case that is not sufficiently fluorescent in an aqueous in vivo liquid medium, and rather becomes
`
`fluorescent in a non-aqueous medium, it is possible to make the pigment fluorescent by allowing the pigment to form
`
`complexes with different living ingredient, (iii) Further, as described below, to introduce a part for uniquely
`
`identifying the detection target to the tracer of the present invention, a complex can be formed with an appropriate
`
`biological ingredient.
`
`[0019]
`
`Actually, the above-described indocyanine pigment does not present any problem in terms of toxicity, but is
`
`substantially non-fluorescent in aqueous solution. Therefore, as shown in the embodiment of the present invention,
`
`indocyanine pigment is enabled to form a complex of the indocyanine pigment and a high-density lipoprotein that is a
`
`biological component having a fat-soluble ingredient, thereby made to contain indocyanine pigment which is made
`
`fluorescent.
`
`[0020]
`
`Furthermore, the near-infrared ray tracer of the present invention is preferably made to be a complex of the
`
`above-described pigment and has the part that uniquely identifies the detection target. The identifying part is
`
`acceptable if it is capable of uniquely identifying the detection target, but otherwise there are no particular
`
`restriction. For instance, identification based on the well-known interactions between proteins, such as the antigen-
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`antibody binding or receptor-ligand binding etc can be used. Moreover, there are no particular restrictions on the
`
`detection target but cells in various living bodies (such as cancer cells) can be used. There are no particular
`
`restrictions about the way the identifying part is provided but, for example, a bond formation reaction used in normal
`
`chemical modification reactions involving proteins is favorably used. In the above-described case, the tracer is
`
`configured to contain a fluorescent pigment, and to have the identification part that uniquely identifies such detected
`
`target.
`
`[0021]
`
`(Imaging using the near-infrared ray tracer) The near-infrared ray tracer of the present invention is capable of
`
`dissolving in vivo liquid medium, and is fluorescent in the near-infrared ray region. Hence, the tracer is introduced
`
`into the living body, and the tracer migrates within the living body through diffusion or the flow of body fluids etc.
`
`It will, therefore, be possible to image in real time the location and concentration change of the tracer by
`
`observing the fluorescence based on the tracer externally from the living body.
`
`[0022]
`
`For example, by chemically bonding anti-tumor antibodies to an indocyanine green - high-density lipoprotein (ICG-
`
`HDL) complex the real-time external measurement of a position and size of a tumor in vivo is enabled. Since such
`
`measurements can be made in real-time using small- scale imaging devices at the time, the imaging method can be used
`
`during surgical dissection of tumors. At the time, tumor tissue can be distinguished from normal tissue in situ, and
`
`leaving tumor tissue behind and doing wasteful excision of healthy tissue will be no longer done.
`
`[0023]
`
`Moreover, since the antibodies to other surface antigens (such as the receptor proteins extending from surface
`
`tissue or the proteins on the outer surface of viruses) can be used as a marker, it is possible to find that where any
`
`antigen, not just limiting to the tumors is located in a living body, and which tissue is infected with a virus can be
`
`easily and uniquely identified. The present invention is thus usable for a diagnostic reagent and diagnostic method for
`
`a variety of diseases.
`
`[0024]
`
`For example, with respect to the image diagnosis of the flow of neuronal axons, if the ICG-HDL complex described
`
`above is administered to the vicinity of the myoneural junction, it will be incorporated into nerve cells and carried
`
`in the flow of neuronal axons. It is possible to observe the determination of the flow of neuronal axons by measuring the
`
`thus- obtained fluorescence extracorporeally
`
`[0025]
`
`When using the tracer described above of the present invention, there are no particular limitations to the device
`
`for measuring the fluorescence from the tracer extracorporeally. The optimal excitation light is irradiated to the
`
`outside of the sample living body with an ordinary excitation light source and if necessary a filter for the excitation
`
`light source, thus a fluorescence detector detects the fluorescence from the pigment that results from the excitation
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`light. If necessary then, a filter can be used to select only the fluorescence from the pigment. It is also preferable
`
`to have a device that does imaging by data processing the obtained fluorescent information. There are no particular
`
`restrictions on the data processing device. One suitable device is the ''ARGUS 20 (Hamamatsu Photonics K.K.) ".
`
`[0026]
`
`By chemically bonding the tracer of the present invention, such as the ICG- HDL, with the anti-tumor antibodies,
`
`it is possible to use the above described imaging method to perform ex vivo measurements of the position and size of a
`
`tumor. If this method is used for instance during surgery, by accurately distinguishing tumor tissue from normal
`
`tissue, the technique can be applied to developing the device to prevent wastefully cut off of the normal tissue or
`
`prevent tumor tissue from being cut and leftover. At this time, to enable bond antibody to ICG-HDL, as explained
`
`already, there are possible methods such as a method to bond HDL part and antibody (cross linking reaction among
`
`proteins, such as cross linking the inter normal amino groups with glutaraldehyde etc.), or a method etc. that cross links ICG parts to amino group of
`
`antibodies.
`
`[0027]
`
`[Embodiments]
`
`As described below, a complex (ICG-HDL) between the near-infrared ray fluorescent pigment indocyanine green (ICG)
`
`and human high-density lipoprotein (HDL) was prepared for use. As used here, HDL is a protein component in blood that
`
`bonds lipids and proteins used here. As described above, ICG is dissolved in the lipid part of the HDL as described
`
`above with the result of becoming fluorescent. The obtained complex is water-soluble; therefore, the ICG-HDL can be
`
`administered to different parts within the living body, and its near-infrared ray fluorescence is to be observed
`
`extracorporeally.
`
`[0028]
`
`Since ICG is already being administered to human bodies in clinical test, and the HDL is originally a living
`
`ingredient, toxicity will be low when the complex is administered to the human body from outside.
`
`[0029]
`
`(1) 20.5 mg of ICG (Diagnogreen, made by Daiichi Pharmaceuticals) was dissolved in 4 ml of distilled water to
`
`prepare an aqueous solution of approximately 5.1 mg/ml (=5.54 mM).
`
`[0030]
`2.5µl of ICG aqueous solution was added to 250 µl of human high-density lipoprotein solution
`
`(HDL, made by Kappel, 20 mg/ml) , and the mixture was stirred to prepare the pigment-protein complex (ICG-HDL).
`
`The ICG concentration in this complex solution was approximately 55.4 µM.
`
`[0031]
`
`(2) The optical characteristics of ICG-HDL,
`
`[0032]
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`The ICG-HDL described above was diluted by a factor of 50 using phosphate buffered saline (PBS), and
`
`a fluorescent spectrum (unadjusted) was measured. For comparison, a spectrum of an organic solution (DMSO)
`
`of the same concentration was also measured. The results are shown in Figure 1. (Note: this is an unadjusted
`
`spectrum, and fluorescence peak values do not match values in the literature (A. Schneider, A. Kaboth, L. Neuhauser:
`
`Detection of sub retinal neovascular membranes with indocyanine green and an infrared ray scanning laser ophthalmoscope,
`
`American.J, of Ophthalmol., 113-1, 45/51 (1932)),
`
`[0033]
`
`From the results, it is clear that the ICG-HDL complex has a similar fluorescent spectrum to the DMSO
`
`solution. That is, the strength of fluorescence of the ICG-HDL complex, was 58.3% (wavelength 835 nm) against the
`
`DMSO solution.
`
`[0034]
`
`(3) Near-infrared ray fluorescent imaging using ICG-HDL.
`
`[0035]
`
`The ICG-HDL obtained above was actually administered to a live experimental animal. The near-infrared ray
`
`fluorescence of ICG was imaged, and the extracorporeal measurement was carried out regarding the way the ICG-HDL was
`
`being distributed within the body. Fig. 2 shows the outline of the measuring system used in the present embodiments.
`
`experimental animal 3, a bandpass filter (with a wavelength centered at 720 nm) was fitted to a 150 W halogen lamp to
`
`prepare an excitation light source 1; and this light was irradiated to the experimental animal 3 through an optical fiber
`
`2. A CCD camera 6 (C2400-75i manufactured by Hamamatsu Photonics Co. Ltd.) fitted with a TV lens (FUJTNGN CF9A1:1.8/8)
`
`was used to detect the near-infrared ray fluorescence of the ICG-HDL to be emitted. However, the infrared cut filter was
`
`removed from the CCD camera and a sharp cut filter (with a transparent wavelength of not less than 840 nm) was set on
`
`the TV lens. Signals from the CCD camera 6 were taken up by an image-processing device 7 (ARGUS20 manufactured by
`
`Hamamatsu Photonics Co. Ltd.)
`
`[0036]
`
`Specifically, a Wister rat on the third day after birth (male) was injected in the right brain with 25 µi of an
`
`ICG-HDL solution through a syringe. Fig. 3 shows its reflected light image (without the sharp cut filter) and its near-
`
`infrared ray fluorescence image (with the sharp cut filter).
`
`[0037]
`
`Fig. 3 is a reflection of the head immediately after administration (fluorescence image with an exposure time of
`
`one second by the CCD camera). It is found that the ICG-HDL was distributed in the forehead centering on the site of
`
`administration. Fig. 4 is a reflection of the head at one hour after administration (exposure time of one second). It is
`
`found from the fluorescence images that the distribution of ICG-HDL was migrating to the occiput. Fig. 5 is a reflection
`
`of the whole body at eight hours after administration (the right part corresponding to the head and the left part
`
`corresponding to the tail with the exposure time of eight seconds). It is found that the
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`ICG-HDL migrated to the terminus of the spinal cord. Further, Fig. 6 shows a schematic representation of the results
`
`from the time-dependent changes discussed above in order to clarify the relationship between the intensities of the
`
`obtained fluorescence and time.
`
`.
`
`[0038]
`
`These results clearly display that the time-dependent observation can be made in a live experimental animal about
`
`the manner how the ICG-HDL migrates from the brain to the spinal cord, from the body surface to a deeper part. This
`
`also shows that the ICG-HDL can be used as a tracer for the observation of a living body sample having thickness.
`
`[0039]
`
`[Effects of the Invention]
`
`The tracer configuration of the present invention, that is, a complex of a near-infrared ray region fluorescent
`
`pigment and an appropriate living body substance, is capable of dissolving in vivo liquid. Further, a pigment that is
`
`low in toxicity but not generally usable as a fluorescent pigment when in aqueous solution, is enabled to be fluorescent
`
`in aqueous solution. Hence, it will become possible to carry out imaging by extracorporeal measurement by using the
`
`tracer according to this invention together with a fluorescence detection device (an excitation light source, detection
`
`system such as CCD and image-processing device).
`
`[0040]
`
`Specifically, there is no need for an X-ray source, laser light source, tomographic detector, high-speed
`
`computer, and the like; yet, the equivalent imaging will be possible. Further, because of convenience and
`
`inexpensiveness, the application in the real time imaging (e.g., during surgery) will also be possible.
`
`[0041]
`In addition, although application sites are restrictive in the angiography using ICG alone, there will be no
`
`such restriction if the ICG-HDL is employed.
`
`[Brief Description of the Drawings]
`
`[Figure 1]
`
`Figure 1 shows the fluorescence spectrum of indocyanine green pigment in DMSO, and the fluorescence
`
`spectrum of the indocyanine - high-density lipoprotein complex in the aqueous solution of the present invention.
`
`[Figure 2]
`
`Figure 2 is a schematic of measurement system for imaging that uses the indocyanine - high-density lipoprotein
`
`complex of the present invention as a tracer.
`
`[Figure 3]
`
`Figure 3 is a photograph showing a reflected light image (top) and near- infrared fluorescent image (bottom)
`
`of a rat immediately after administering ICG-HDL.
`
`[Figure 4]
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`Figure 4 is a photograph showing a reflected light image (top) and near- infrared fluorescent image (bottom)
`
`of the rat 1 hour after administering the ICG-HDL.
`
`[Figure 5]
`
`Figure 5 is a photograph showing a reflected light image (top) and near- infrared fluorescent image (bottom)
`
`of the rat 8 hours after administering the ICG-HDL.
`
`[Figure 6]
`
`Figure 6 is a schematic showing the result of change over time and a relationship between the time and obtained
`
`strength of fluorescence after administering the ICG-HDL to the rat.
`
`[Description of Symbols]
`1 ... Excitation light source
`
`2 ... Optical fiber
`
`3 ... Test subject (newly born rat)
`
`4 ... ICG-HDL tracer
`
`5 ... Sharp cut filter (840 nm)
`
`6 ... Detector (camera)
`
`7
`
`8
`
`... Image processor
`
`…... Fluorescence
`
`[Procedure Amendment Document]
`
`[Filing Date] August 15, 1996 [Amendment 1]
`
`[Document for Amendment] Specification [Item for Amendment] Figure 3
`
`[Method of Amendment] Change
`
`[Contents of Amendment]
`
`[Figure 3]
`
`Figure 3 is a microscope photograph showing a reflected light image (top) and near-infrared ray fluorescent
`
`image (bottom) of a rat immediately after administering ICG-HDL.
`
`[Amendment 2]
`
`[Document for Amendment] Drawings [Item for Amendment] Figure 4
`
`(Method of Amendment] Change [Contents of Amendment]
`
`[Figure 4] Figure 4 is a microscope photograph showing a reflected light image (top) and near-infrared ray fluorescent
`
`image (bottom) of a rat one hour after administering ICG-HDL.
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`Fig. 2
`
`MEASURING SYSTEM SCHEMATIC
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`Fig.4
`
`REFLECTED IMAGE (TOP) AND NEAR-
`INFRARED RAY FLUORESCENT IMAGE (BOTTOM)
`OF A RAT 1 HOUR AFTER ADMINISTERING ICG-
`HDL
`
`PHOTOGRAPHS IN PLACE OF DRAWINGS (COLOR)
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`Fig. 6
`
`(A
`)
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`Fig.
`1
`
`Fig. 6
`
`[illeg]
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`Wave length mn
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`Fig 3
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`REFLECTED IMAGE (TOP) AND NEAR-INFRARED RAY FLUORESCENT IMAGE (BOTTOM) OF A RAT 1 HOUR AFTER ADMINISTERING ICG-HDL
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`PHOTOGRAPHS IN PLACE OF DRAWINGS (COLOR
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`Fig. 5
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`REFLECTED IMAGE (TOP) AND NEAR-INFRARED RAY FLUORESCENT IMAGE (BOTTOM) OF A RAT 8 HOUR AFTER ADMINISTERING ICG-HDL
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`PHOTOGRAPHS IN PLACE OF DRAWINGS (COLOR
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`Reflected light image (top) and near-infrared ray image (bottom) administered with ICG-HDL
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`Right after administration
`
`[Procedure amendment 2]
`[Target amendment document] drawing
`[Target amendment item No] Fig. 4
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`Picture in place of drawing
`
`[Amendment method] Change
`[Amendment content] Fig. 4
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`Reflected light image (top) and near-infrared ray image (bottom) one hour after administered
`
`Procedure amendment 3]
`[Target amendment document] drawing
`[Target amendment item No] Fig. 5
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`Picture in place of drawing
`
`[Amendment method] Change
`[Amendment content] Fig. 5
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`Reflected light image (top) and near-infrared ray image (bottom) eight (8) hours
`after administered
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`Picture in place of drawing
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