JPH0783917A - Dying coloring matter for inspecting light echo and optical inspection method - Google Patents

Abstract

PURPOSE:To accurately detect a microstructure change of a biological sample by using a dying coloring matter for inspecting light echo with a specific chemical structure normally called rhodamine 640 system. CONSTITUTION:By using a dyeing coloring matter for inspecting light echo which has a chemical structure expressed by an expression I and selects either one of two tapes of -COO and -SO3 for X in the expression I, nothing is added to Y or either one of five types of -SO2C, -N=C=S, expression II, expression III, and expression IV is selected for Y, a biological sample is dyed. Then, light echo from the living thin sample is measured by a laser-scanning microscope type light echo microscope and either normal tissue of abnormal tissue is judged according to the difference in phase relaxing time, thus accurately detecting the microstructure change of the biological sample.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dye for an optical inspecting device used for optically observing a difference in physicochemical properties of substances and an optical inspecting method using the dye.

[0002]

2. Description of the Related Art Generally, there is known a laser scanning microscope for observing a sample from optical information obtained by scanning the sample with a laser beam. However, the information of the sample obtained by the conventional laser scanning microscope is qualitatively the same as the information obtained from the image of an ordinary optical microscope.

On the other hand, the applicant of the present invention discloses in Japanese Patent Laid-Open No. 4-26.
In 9644, a photo-echo microscope was proposed as a microscope capable of obtaining information regarding a difference in physicochemical properties of a sample, which cannot be obtained by an ordinary optical microscope.
The optical echo microscope irradiates a sample with laser light, detects a light echo in a light absorber that can resonate with the laser light contained in the sample, and detects the optical phase relaxation time of this light echo (generally, It is a time called “T ₂ relaxation time” or “transverse relaxation time”). The phenomenon of optical echo is described in, for example, “Ultrafast optical technology” (edited by Tatsuo Yajima, Maruzen, published in March 1990).

In Japanese Unexamined Patent Publication (Kokai) No. 4-269644, a combination of optical echo measurement and a laser scanning microscope is used, and the laser beam and the sample are two-dimensionally and relatively three-dimensionally relatively scanned. It was stated that the distribution state of the physicochemical properties of the sample can be clarified by mapping the obtained phase relaxation time in two dimensions or three dimensions. Further, at the same time, an image as a normal laser scanning microscope can be obtained.

For example, when a dye that can resonate with laser light is dispersed in a polymer in which crystalline and amorphous are mixed,
By utilizing the fact that the optical phase relaxation time differs between crystalline and amorphous, it is possible to determine how the crystalline phase and the amorphous phase are mixed in the polymer by optical echo. It can be observed under a microscope. In the case of a solid, its optical phase relaxation time can be easily measured by the heterodyne detection method of accumulated photon echo by intensity modulation. Further, a heterodyne detection method of accumulated photon echo by optical phase modulation can also be used. The heterodyne detection method by optical phase modulation of optical echo is disclosed in Japanese Patent Application Laid-Open No. 4-1322020 by the present applicant. Hereinafter, the measuring method will be described in JP-A-4-132020.

The measurement in JP-A-4-132020 mainly uses pulsed light, and basically utilizes a phenomenon called stimulated photon echo (induced optical echo) in photon echo (optical echo). As is well known, the photoexcited state of a substance is the equation of motion (Liovil) of its density matrix.
Le's equation). For convenience, the relaxation time of the diagonal component of the density matrix is called T ₁ hour (longitudinal relaxation time), and the relaxation time of the non-diagonal component is called T ₂ hour (horizontal relaxation time). Different. Longitudinal relaxation is considered to mean the process in which the photoexcited state relaxes with the release of energy, and transverse relaxation refers to the disturbance of the coherency of the oscillation of electrical polarization brought into a substance by incident light. It is believed to show the process of going.

The phenomenon called optical echo (photon echo) is considered to be a kind of third-order nonlinear optical effect, and the induced photon echo (induced optical echo) therein will be described with reference to FIG. Consider the case where a substance is excited with an appropriate pulsed light in energy resonance. First, the pulsed light of E ₁ is irradiated at t ₁ , and then the pulsed light of E ₂ is irradiated at t ₂ . Next, when irradiated with t ₃ at a third pulse _{E 3, (t 3 + t} 2 -t 1 = t 3 + τ (τ = t 2 -
At the time of t ₁ )) (τ: delay time), light is emitted from the substance. This is the stimulated photon echo light.
The one that emits stimulated photon echo light by irradiating three pulses in this way is called a three-pulse echo. The intensity of the stimulated photon echo light is exp (-4τ / T ₂ ).
Decays in proportion to. Further, when the heterodyne detection method by optical phase modulation is used, the intensity of the stimulated photon echo light is attenuated in proportion to exp (−2τ / T ₂ ). Therefore, in the optical echo microscope disclosed in JP-A-4-132020, the intensity of the stimulated photon echo light (particularly the accumulated photon echo light) is measured while changing τ, so that T ₂ (transverse) different for each substance or each state can be obtained. Relaxation time).

When the above-mentioned Lioville equation is calculated by perturbation expansion by the rotating wave approximation and the weak excitation light approximation, a third-order nonlinear polarization wave, that is, an echo wave is obtained. The photon echo is a kind of third-order nonlinear optical phenomenon, and its effect is generally small. Regeneration excitation light (E ₃ )
On the other hand, the photon echo light intensity is usually several orders of magnitude weaker. It is known that detection of such weak light (photon echo light) is improved by utilizing interference of light.

In this case, weak light (photon echo light)
Is observed (measured) by interfering with a light wave (probe light E ₄ ) having a known phase characteristic, amplifying a change in intensity and phase of weak light (photon echo light). At this time, the intensity or frequency of the weak light (photon echo light) is modulated by some method, and only the synchronous component in the output of the combined light (the combined light of the photon echo light and the probe light) is amplified to increase the contrast. Is commonly practiced.

In JP-A-4-132020, the following is done. _{t 43 -t 21 (t 21 =} t 2 -t 1, t
It can be seen that when ₄₃ = t ₄ −t ₃ ) is modulated for one or more light cycles, the intensity of the combined light of the echo light and the probe light is also modulated in synchronization with it. Here, it is considered that t _43, that is, the delay time between the excitation light and the probe light (the delay time at the time of reading) is synchronized and oscillated. This is equivalent to phase-modulating the probe light relative to the excitation light. At this time, it can be seen that only the modulation component having an even multiple of the phase modulation frequency remains.

Therefore, the excitation light and the probe light are phase-modulated relatively at the frequency f, the combined light of the echo light and the probe light is detected by the photodetector, and f is converted from the electric signal obtained by photoelectric conversion. It is possible to stably detect the echo light with high accuracy by extracting only even-numbered (2 times, 4 times, 6 times ...) Components. In the experimental example described in JP-A-4-132020, the light source serves both as an excitation light source and a probe light source, is a harmonic excitation Kiton red dye laser of a mode-locked YAG laser, and has a repetition frequency of 82 MHz. The pulsed light of is output. All wavelength selective elements were removed from the Kiton Red dye laser. As a result, the central wavelength is 620 nm and the spectral width is 400 cm ^-1.
Incoherent light with a correlation time of 37 femtoseconds was obtained. Also, t ₁ , t ₂ , and
FIG. 8 shows the relationship between t ₃ and t ₄ . t ₁ and t ₃ , t ₂ and t
₄ is repeated at 82 MHz, t ₁ and t ₂ , t ₃ and t ₄
During this period, phase modulation is applied by the phase modulation means.

As described above, the optical echo microscope optically observes the difference in the physicochemical properties of substances.

[0013]

However, in the above-mentioned optical echo microscope, when a biological sample is inspected, the dyes are not limited, and it is unsuitable for detecting microstructural changes in the biological sample. Sometimes used different dyes. When an inappropriate dye is used, there is a problem that it is not possible to accurately detect a microscopic structural change in a biological sample.

In view of the above point, the present invention has an object to provide a dye for optical echo inspection and an optical inspection method capable of accurately detecting microscopic structural changes in a biological sample.

[0015]

As a result of research in the present invention,
It was found that the microscopic structural change of a biological sample can be detected by using the dye for photoecho examination described in (claim 1). These dyes are usually rhodamine 640
It is called a dye, a rhodamine X dye, or a Texas red dye.

Therefore, the present invention firstly provides a dye for optical echo inspection as a dye for optical echo inspection, which is used when "inspecting a biological sample by measuring the optical echo of the stained biological sample, The dye for optical echo inspection has a chemical structure represented by the following general formula [A],

[0017]

[Chemical 3]

[0018] The X of the [A] ^-COO -, -SO ₃
^- any one Two of selected of, or not stick anything to _{Y, -SO 2 Cl, -N =} C = S,

[0019]

[Chemical 4]

Any one of the five types is selected (Claim 1). Secondly, the optical inspection method is "in an optical inspection method for inspecting a biological sample by measuring an optical echo of the stained biological sample,
The dye for staining the biological sample is the dye for optical echo inspection according to claim 1 (claim 2).

[0021]

By using the dye for optical echo examination of the present invention (hereinafter referred to as a dye), it is possible to accurately detect a microscopic structural change in a biological sample. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0022]

FIG. 7 is a schematic block diagram of an optical inspection apparatus used in the first embodiment of the present invention. As shown in this figure, the optical system of this example is roughly divided into two parts, each surrounded by a dotted line, that is, a light source means (A) and a light modulation optical system (B). The light source means (A) of this example is the laser light source 1 itself, and the laser light source 1 is
It is a dye laser excited by a mode-locked argon ion laser of Hz. Besides, as the laser light source 1, a multi-mode semiconductor laser, a mode-locking semiconductor laser, a light emitting diode or a solid-state laser can be used. Here, it is preferable that the laser light source 1 has a center wavelength on the longer wavelength side than the wavelength having the maximum absorption coefficient of the light absorption band of interest for the object to be inspected. Further, the light emitted from the laser light source 1 may have a spatial coherence that can be regarded as a point on the image plane and a coherence that is shorter than the optical phase relaxation time of the light absorption band of interest of the object to be inspected. preferable.

In FIG. 7, the laser light emitted from the laser light source 1 is incident on the input side optical splitter 2 of the optical modulation optical system (B), and the optical splitter 2 splits the laser beam into two light beams. To do. On the output side of the light modulation optical system (B), an optical multiplexer 3 that multiplexes the two light beams thus branched is arranged. Although both the optical splitter 2 and the optical multiplexer 3 are polarization beam splitters, a semi-transparent mirror having little or no polarization characteristics can be used instead.

The first orthogonal reflection surfaces 6a and 6b and the second orthogonal reflection surface 7 are arranged in parallel between the optical splitter 2 and the optical multiplexer 3.
a and 7b are arranged. As the optical system that divides the light from the light source means (A) into two and combines them again, a two-path interference optical system similar to a Michelson interferometer or a Mach-Zehnder interferometer is suitable. The light beam that has passed through the split surface of the light splitter 2 is reflected by one of the first orthogonal reflecting surfaces 6a and then travels toward the first corner cube 4a fixed to a movable stage (not shown). This corner cube 4
The light beam reflected by a is reflected by the other reflecting surface 6b of the first orthogonal reflecting surface and enters the optical multiplexer 3. Reference numeral 4b denotes a displacement device that displaces the corner cube 4a via the movable stage. An optical delay unit 4 is composed of the corner cube 4a, the movable stage and the displacement unit 4b. By moving the corner cube 4a using the displacement device 4b, it is possible to delay one of the light fluxes by a predetermined amount. Here, instead of the corner cube 4a, two mirrors or a right angle prism may be used.

The light beam reflected by the split surface of the light splitter 2 is
After being reflected by one of the reflecting surfaces 7 a of the second orthogonal reflecting surfaces, it enters the phase modulating means 5. As the phase modulation means 5, generally one using an electro-optic crystal is well known, but in the first embodiment, a piezoelectric element 5b having one end fixed and a corner cube 5a fixed at the other end is used. ing. Then, the piezoelectric element 5b is driven by the AC driving power source 5c.
An alternating voltage of a predetermined frequency f is applied to the phase modulation means of the frequency f. Where corner cube 5
Instead of b, a rectangular prism having a small dispersion may be used. The light flux that has passed through the phase modulation means 5 is reflected by the second orthogonal reflection surface 7b and enters the optical multiplexer 3. In the first embodiment, the optical delay means 4 is provided on the transmission optical path of the optical splitter 2.
Although the phase modulation means 5 is arranged on the reflected light path, the invention is not limited to this, and the opposite arrangement is possible, and both the optical delay means 4 and the phase modulation means 5 can be arranged on the same light path. is there. An optical modulation optical system (B) is configured by the optical splitter 2, the optical multiplexer 3, the optical delay means 4 and the phase modulation means 5. The two light fluxes in the light modulation optical system (B) are combined by an optical combiner 3 and passed through an appropriate lens system 8 to the sample chamber 2
The biological sample 20 in 1 is irradiated. The lens system 8 can also be used in common with the objective optical system of the optical microscope. In that case, if an eyepiece optical system is additionally provided, it becomes an optical microscope, and visual observation is also possible. Further, by combining an irradiation optical system and another confocal optical system, arranging a photodetector with a pinhole in front, and combining the sample chamber 21 with a movable stage, a laser scanning microscope type optical echo microscope is obtained. . The sample chamber 21 can be cooled if necessary.

The detector 10 composed of a photomultiplier tube is arranged so that light emission due to fluorescence or phosphorescence in the light spot forming portion of the biological sample 20, reflection, diffraction or scattering of irradiation laser light can be detected well. A filter is inserted in front of the detector 10 if necessary. Here, the detector 1
The position of 0 is not limited to the position shown in the figure, and may be an arrangement for detecting transmitted light.

The detector 10 outputs a signal corresponding to the amount of incident light by photoelectric conversion, and of the signals output from the detector 10, only the modulation component having twice the phase modulation frequency is amplified by the lock-in amplifier 11. It The amplified signal is stored and analyzed in the data processing device 50 via the electric signal processing device 40 according to the delay time of the optical delay device 4.

The inspection is performed using the optical inspection device as described above. X as the staining dye formula as in claim 1 [A] is -COO ^-, Y is -N = C = S XR
Using ITC, normal and tumor tissues (abnormal tissues) of human pancreas are used as biological samples. The biological sample was stained with XRITC and examined. The temperature of the sample was 6 Kelvin. As an experimental condition, the phase modulation frequency of the phase modulator 5 was set to 20 KHz. When only the 40 KHz electric signal is amplified and recorded by the lock-in amplifier 11 while scanning the stage position of the optical delay device 4, an optical phase relaxation time is obtained, and an objective inspection result can be obtained based on the information. It was On the long wavelength side of the absorption band having a peak at about 580 nm of XRITC, the center wavelength of the laser is about 600.
When measured in nm, high S /
The optical echo could be measured at the N ratio. The relationship between the intensity I of the optical echo, the delay time τ, and the phase relaxation time T ₂ is expressed by the following equation (1).

I = I ₀ exp (−2τ / T ₂ ) I ₀ :
Optical echo intensity when τ = 0 ∴T ₂ = −2τ / ΔlogI (1) (logI−logI ₀ = ΔlogI) FIG. 1 shows normal tissues of human pancreas according to the first embodiment of the present invention. the differences in tumor tissue is a diagram examined from the difference phase relaxation time T _2.

FIG. 1 is a diagram showing the intensity I of the optical echo with respect to the delay time τ, and the phase relaxation time T ₂ can be obtained from this FIG. 1 and the equation (1). The phase relaxation time T ₂ obtained as described above is 47 ps for the normal tissue and 2 for the tumor tissue.
It was 9 ps. It was possible to clearly distinguish the normal tissue and the tumor tissue from the obtained phase relaxation time T ₂ . It can be seen that the tumor tissue has a shorter phase relaxation time T ₂ than the normal tissue. That is, it was possible to distinguish between normal and abnormal human pancreas, and a highly sensitive pathological examination was possible. The biological sample was not limited to the pancreatic tissue sample, but it was also possible to distinguish normal from abnormal in tissues of other sites.

2 and 3 are diagrams in which the difference between normal tissue and tumor tissue of human pancreas according to the second embodiment of the present invention is examined from the difference in phase relaxation time T ₂ . FIG. 2 shows an examination of normal tissue, and FIG. 3 shows an examination of tumor tissue (abnormal tissue). The inspection is performed using the same optical inspection device as in the first embodiment. Further, X is -SO ₃ of the general formula according to claim 1 as a dye for staining [A] ^-, Y is -S
Texas Red, which is O ₂ Cl, was used. Normal and tumor tissues (abnormal tissues) of human pancreas were stained with this Texas red and examined.
As shown in, the optical echo could be measured at a high S / N ratio. 2 and 3 are also diagrams showing the intensity I of the optical echo with respect to the delay time τ similarly to FIG. 1, and the phase relaxation time T ₂ is obtained from these FIGS. 2 and 3 and the equation (1). The phase relaxation time T ₂ thus obtained is 54.8 for normal tissue (FIG. 2).
Tumor tissue (Figure 3) was 34.8 ps at ps. It was possible to clearly distinguish the normal tissue and the tumor tissue from the obtained phase relaxation time T ₂ . It can be seen that the tumor tissue has a shorter phase relaxation time T ₂ than the normal tissue.
That is, it was possible to distinguish between normal and abnormal human pancreas, and a highly sensitive pathological examination was possible. The biological sample was not limited to the pancreatic tissue sample, but it was also possible to distinguish normal from abnormal in tissues of other sites. The other points are the same as in the first embodiment.

FIG. 4 shows the phase relaxation time T depending on the presence or absence of an antigen-antibody reaction of human laryngeal cancer-derived cells according to the third embodiment of the present invention.
It is the figure inspected from the difference of ₂ . Human-derived antinuclear antibody (antibody) to human laryngeal cancer-derived cells (HEp-2) (antigen)
X in the general formula [A] of claim 1 with a sample not reacted with reacted sample as a staining dye is -COO ^-,
Y is

[0033]

[Chemical 5]

Rhodamine X maleimide (XRM)
When it was dyed with and was inspected with the same optical inspection apparatus as in the first embodiment, the optical echo could be measured with a high S / N ratio as shown in FIG. FIG. 4 is also a diagram showing the intensity I of the optical echo with respect to the delay time τ, and the phase relaxation time T ₂ can be obtained from this FIG. 4 and the equation (1). Further, the obtained phase relaxation time T ₂ is 50 for the sample which has not reacted with the antigen-antibody reaction.
The sample reacted with the antigen-antibody at ps was 39 ps.

The presence or absence of the antigen-antibody reaction could be clearly distinguished from the obtained phase relaxation time T ₂ . It can be seen that the sample in which the antibody reacts has a shorter phase relaxation time T ₂ than the sample in which the antibody does not exist. That is, the presence or absence of an antigen-antibody reaction can be distinguished, and a highly sensitive pathological examination was possible. As a biological sample, the presence or absence of an antigen-antibody reaction could be confirmed not only in cells derived from human laryngeal cells but also in cells at other sites.

Others are the same as in the first embodiment.
5 and 6 are diagrams in which the presence or absence of an antigen-antibody reaction of human laryngeal cancer-derived cells according to the fourth embodiment of the present invention is examined from the difference in phase relaxation time T ₂ . FIG. 5 is a sample that has undergone an antigen-antibody reaction, and FIG. 6 is a sample that has not undergone an antigen-antibody reaction.

A general formula [A] described in claim 1 in which a sample obtained by reacting human laryngeal cancer-derived cells (HEp-2) (antigen) with a human-derived antinuclear antibody (antibody) and a sample not reacted are used as staining dyes. X of] is -COO ^-, Y is

[0038]

[Chemical 6]

When it was dyed with XRIA and was inspected by the same optical inspection apparatus as in the first embodiment, the optical echo could be measured at a high S / N ratio as shown in FIGS. . 5 and 6 are also diagrams showing the intensity I of the optical echo with respect to the delay time τ, and the phase relaxation time T ₂ can be obtained from these FIGS. 5 and 6 and the equation (1). Further, the obtained phase relaxation time T ₂ is 64.8 ps for the sample (FIG. 6) which has not undergone the antigen-antibody reaction and 5 for the sample which has undergone the antigen-antibody reaction (FIG. 5).
It was 1.9 ps.

The presence or absence of an antigen-antibody reaction could be clearly distinguished from the obtained phase relaxation time T ₂ . It can be seen that the sample in which the antibody reacts has a shorter phase relaxation time T ₂ than the sample in which the antibody does not exist. That is, the presence or absence of an antigen-antibody reaction can be distinguished, and a highly sensitive pathological examination was possible. As a biological sample, the presence or absence of an antigen-antibody reaction could be confirmed not only in cells derived from human laryngeal cells but also in cells at other sites.

The other points are the same as those in the first embodiment.
It should be noted that, in the case of the staining dyes of the combination of X and Y of the general formula [A] described in claim 1 other than the staining dyes described in the first to fourth examples, the normal tissue and the abnormal tissue or the antigen-antibody reaction are similarly obtained. I was able to distinguish the presence or absence of.

[0042]

EFFECTS OF THE INVENTION By using the dye for optical echo examination of the present invention, it becomes possible to accurately detect a microscopic structural change in a biological sample. Further, according to the optical inspection method of the present invention, it is possible to accurately detect a microscopic structural change in a biological sample.

[Brief description of drawings]

FIG. 1 is a diagram showing a difference in phase relaxation time between normal tissue and tumor tissue of human pancreas stained with XRITC according to the first embodiment of the present invention.

FIG. 2 is a diagram showing the phase relaxation time of normal tissue of human pancreas stained with Texas Red according to the second embodiment of the present invention.

FIG. 3 shows the phase relaxation time of human pancreatic tumor tissue stained with Texas Red according to the second embodiment of the present invention.

FIG. 4 is a diagram showing the presence / absence of an antigen-antibody reaction according to the third embodiment of the present invention based on the difference in phase relaxation time.

FIG. 5 is a diagram showing a phase relaxation time of a biological sample which has undergone an antigen-antibody reaction according to a fourth example of the present invention.

FIG. 6 is a diagram showing a phase relaxation time of a biological sample that has not undergone an antigen-antibody reaction according to the fourth example of the present invention.

FIG. 7 is a schematic configuration diagram showing an optical inspection device preferably used in the first embodiment of the present invention.

FIG. 8 is an explanatory diagram of a principle of measuring an optical echo.

[Explanation of symbols for main parts]

DESCRIPTION OF SYMBOLS 1 ... Light source 2 ... Optical splitter 3 ... Optical multiplexer 4 ... Optical delaying means 5 ... Optical modulating means 8 ... Lens system 10 ... Photodetector 30 ... Control means 40 ... Electrical signal processing device 50 ... Data processing device

Claims (2)

[Claims] 1. A dye for optical echo inspection used for inspecting a biological sample by measuring an optical echo of the stained biological sample, wherein the dye for optical echo inspection is represented by the following general formula: A]
It has a chemical structure represented by In the above [A], one of two kinds of —COO ⁻ and —SO ₃ ^— is selected for X, and nothing is attached to Y.
SO ₂ Cl, -N = C = S, Any one of the five types described above is selected, and a dye for optical echo inspection. 2. An optical inspection method for inspecting a biological sample by measuring an optical echo of the stained biological sample, wherein the staining dye for staining the biological sample is the dye for optical echo inspection according to claim 1. An optical inspection method characterized by being present.