JP2005227155A - Image sensor for measuring biotexture, and biotexture measuring method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To allow an observation for a biotexture of a living organ under the condition where the living organ maintains an intrinsic activity condition, and to obtain a high grade of biological information by combination of an observation of electric stimulation or a bioelectric signal and an observation of light emission/fluorescence phenomenon in the biotexture. <P>SOLUTION: A plurality of protrusion electrodes 16 is provided on a substrate 10 with a prescribed separate space to expose a large number of picture element cells 13 from a protection layer coating an upper face of a photoelectric transfer area 11 arranged two-dimensional-arrayedly. The protection layer comprises a resin not injuring the biotexture, the biotexture is placed directly on the protection layer, or the sensor itself is embedded in the biotexture, and an emission light from the biotexture is thereby received by a photodiode 14 very close thereto. Since the each protrusion electrode 16 contacts with the biotexture, a very weak current is made to flow in the biotexture via the protrusion electrode 16 to be simulated electrically, an image due to optical reaction thereto is observed, or an optical phenomenon and an electric phenomenon by the biotexture are measured at the same time. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention is mainly for measuring biological tissue suitable for measuring and observing various biological tissues such as biological cells, proteins, DNA, peptides, saccharides in fields such as biochemistry, molecular biology, and clinical medicine. The present invention relates to an image sensor and a biological tissue measurement method using the sensor. The “image sensor” referred to here includes both a solid-state image sensor (semiconductor chip) itself and a sensor unit configured around the solid-state image sensor.

In recent years, as a bioimaging technique, the importance of a technique for observing an optical phenomenon in a living cell has been greatly increased. Examples of such techniques include luminescence observation using photoproteins such as aequorin and house taurine, fluorescence observation using GFP (Green Fluoresence Protein), Ca ²⁺ ion-sensitive dyes such as Fura-2 and Indo-1, and the like. And cell activity observation using a voltage sensitive dye. All of the current bioimaging techniques using the light emission phenomenon and the fluorescence phenomenon of living tissue are to enlarge and observe the phenomenon generated under a microscope.

  As the microscope, a near-field microscope (total reflection fluorescent microscope) is mainly used (see Non-Patent Document 1, etc.), but such a special microscope has a very complicated optical system, a large apparatus itself, and a low price. Pretty expensive. Therefore, there is a strong demand for the development of an apparatus that can acquire an image due to an optical phenomenon of a living tissue more easily and at a low cost.

  In general, the fluorescence and self-emission of living tissue is quite weak. However, with conventional devices, such weak light is further attenuated while passing through the optical system, and sufficient fluorescent and luminescent images can be obtained. In some cases, it could not be obtained. Moreover, distortion may occur in a part of the fluorescence image / light emission image due to a distortion factor such as aberration of the optical system.

  Furthermore, the observation object that can be observed by an apparatus such as a conventional microscope is limited to a size and a form that can be set on the microscope. Therefore, basically, only living tissue isolated from a living body can be observed. For example, living tissue such as a relatively large animal remains in the living body (that is, without being collected from the animal). B) It was very difficult to observe, and there was a limit to the phenomena that could be observed.

“Arc light source total reflection fluorescence microscope system IX71-ARCEVA”, [online], Olympus Corporation, [searched on January 20, 2004], Internet, <URL: http://www.olympus.co.jp/jp /lisg/bio-micro/product/ix71-arceva.h>

  The present invention has been made in view of these problems, and its main purpose is a two-dimensional image by various optical phenomena such as self-luminous phenomenon, fluorescent phenomenon, or light absorption phenomenon by various biological tissues such as biological cells. It is an object of the present invention to provide a biological tissue measurement image sensor and a biological tissue measurement method that can be obtained easily and at low cost.

  Further, another object of the present invention is suitable for various optical measurement methods conventionally performed for observing the dynamic aspect of a living tissue, and has a high sensitivity and good two-dimensionality. An object of the present invention is to provide a biological tissue measurement image sensor and a biological tissue measurement method capable of acquiring an image.

Means for Solving the Problems and Effects of the Invention

The biological tissue measurement image sensor according to the first invention, which has been made to solve the above-mentioned problems, is a biological tissue measurement image sensor for acquiring a two-dimensional image by a light phenomenon of a biological tissue that is an observation object. And
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) In the region of the photoelectric conversion unit, the upper end of the photoelectric conversion unit is exposed or protruded from the protective film layer, and is used to apply electrical stimulation to the observation object and / or to localize the observation object. An electrode part for detecting a potential,
The observation object is imaged in a state where the observation object is in contact with the protective film layer.

  As one aspect of the biological tissue measurement image sensor according to the first aspect of the present invention, it is preferable that a plurality of the electrode portions are arranged two-dimensionally at a predetermined interval in the region of the photoelectric conversion portion.

  When measurement is performed using the biological tissue measurement image sensor according to the first aspect of the present invention, a biological tissue that is an observation object, such as a biological cell, is directly applied to the protective film layer that covers the surface of the photoelectric conversion unit of the sensor. Arranged to touch. In this state, since the living tissue is in close proximity to the photoelectric conversion unit with the protective film layer interposed therebetween, light emitted from the living tissue (not only fluorescent light and self-emitting light but also the living tissue itself is emitted). Even if the light transmitted through the living tissue is weak, the light reaches the minute light receiving elements arranged in a two-dimensional manner without being attenuated, and is photoelectrically converted. For this reason, the light transmission distance is extremely short and the spread is almost negligible, so that the projected image on the photoelectric conversion unit is almost the same size as the observation object.

  Therefore, if the biological tissue measurement image sensor according to the first invention is used for observation of biological tissue, a highly sensitive and clear image can be obtained. In addition, since a complicated optical system unlike a near-field microscope is not required, imaging can be performed at a much lower cost than in the past. In addition, since the protective film layer in contact with the living tissue does not harm the living tissue, the purpose of the measurement is as follows without inhibiting the activity of the living tissue or damaging the living tissue. It is possible to accurately capture the dynamic aspect of the living tissue. This also applies to biological tissue measuring image sensors according to second to sixth inventions described later.

  In the biological tissue measurement image sensor according to the first aspect of the present invention, when the biological tissue is in contact with the protective film layer, the tip of the electrode portion that is exposed or protrudes from the protective film layer is in contact with the biological tissue. This electrode part can serve two functions. That is, one of them is to apply electrical stimulation to a living tissue by applying a predetermined potential to the electrode part or flowing a predetermined minute current from the electrode part. By combining such electrical stimulation with respect to biological tissue and imaging of optical phenomena of biological tissue, for example, observation of changes in optical phenomena when electrical stimulation is applied to biological tissue, or conversely, a captured image of biological tissue Various forms of measurement are possible, such as applying a predetermined electrical stimulus to a living tissue based on information collected from. In particular, by providing a plurality of electrodes in a two-dimensional manner, it is possible to locally apply electrical stimulation to living tissue, or to change the site to which electrical stimulation is applied over time, and variations in measurement are possible. spread.

  The function of the other electrode part is measurement of an electric signal generated in the living tissue. Thereby, the imaging of the optical phenomenon of the living tissue and the electrical measurement of the living tissue can be performed simultaneously.

  By the way, in a general solid-state imaging device, a method of detecting the amount of photocharge within a certain time as a potential is used when converting the incident light intensity into an electric signal by the micro light receiving device of each pixel cell. In the target imaging, for example, when the light emission intensity is originally very weak, such as when observing a luminescent protein, or when high intensity excitation light is present at the same time as observing the fluorescence of a fluorescently labeled protein. There are cases where photographing under fairly severe conditions is required, such as when fluorescent light has to be detected under such conditions. Under these conditions, it is difficult to ensure a sufficient S / N ratio with a conventional detection method.

In view of these points, the biological tissue measurement image sensor according to the second aspect of the present invention is a biological tissue measurement image sensor for acquiring a two-dimensional image due to a light phenomenon of a biological tissue that is an observation object,
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) a signal conversion means for converting the light intensity signal received by each of the micro light receiving elements into a pulse signal sequence having information on the pulse width or pulse frequency by pulse width modulation or pulse frequency modulation;
The observation object is imaged in a state where the observation object is in contact with the protective film layer.

  That is, in the biological tissue measurement image sensor according to the second aspect of the invention, the light intensity incident on the minute light receiving element is not converted into an analog level electric signal, but the signal conversion means performs either pulse frequency modulation or pulse width modulation. This is converted into a pulse signal sequence of a predetermined height (voltage) and output. Pulse frequency modulation converts the light intensity, which is an analog value, into the frequency of the pulse signal, and is known as an output form in the biological nervous system, and accurately detects changes in light intensity rather than the absolute value of light intensity. be able to. Therefore, it is particularly suitable for the case where minute fluorescent light has to be detected under the condition in which excitation light having a large intensity as described above is simultaneously present. On the other hand, the pulse width modulation is to convert the light intensity, which is an analog value, into a pulse width and output it. Preferably, the circuit is configured so that the pulse width increases as the light intensity decreases. In this configuration, when extremely weak light is incident, a weak light intensity difference is enlarged and detected as a pulse width difference. Therefore, it is particularly suitable when the incident light intensity as described above is very weak. In any case, since the height of the pulse does not depend on the light intensity, it is highly resistant to external noise.

  As described above, the biological tissue measurement image sensor according to the second aspect of the present invention can detect the target incident light intensity and its change with high sensitivity and accuracy even under severe conditions peculiar to the measurement of the optical phenomenon of the biological tissue. can do. Therefore, it is possible to acquire an image that clearly captures an optical phenomenon caused by a living tissue.

A biological tissue measurement image sensor according to a third aspect of the present invention for solving the above-described problems is a biological tissue measurement image sensor for acquiring a two-dimensional image based on a light phenomenon of a biological tissue that is an observation target. Because
a) A large number of micro light receiving elements are two-dimensionally arranged, and each micro light receiving element is formed in a shallow portion inside the substrate and mainly includes a short wavelength detection region for detecting light having a relatively short wavelength, A photoelectric conversion unit having a long wavelength detection region that mainly detects light having a relatively long wavelength formed in a deeper portion inside the substrate than the short wavelength detection region;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) a signal extraction means for independently extracting an electric signal generated by photoelectric conversion in the short wavelength detection region and an electric signal generated by photoelectric conversion in the long wavelength detection region;
In the state where the observation object is in contact with the protective film layer, an electrical signal for light emitted from the observation object at two different wavelengths is transmitted in the short wavelength detection region and the long wavelength detection region. It is characterized by acquiring at the same time.

  The biological tissue measurement image sensor according to the third aspect of the invention is suitable, for example, for one-wavelength excitation / two-wavelength fluorescence ratio measurement. In the one-wavelength excitation / two-wavelength fluorescence measurement, when a biological tissue arranged so as to be in contact with the protective film layer is irradiated with excitation light having a predetermined wavelength, they are different from each other depending on the components of the biological tissue, the activity status, etc. Wavelengths of fluorescent light can be emitted. The fluorescence emitted from the living tissue may be a mixture of two different wavelengths (peak top wavelengths). When such fluorescence enters the micro light receiving element, light having a relatively long wavelength component penetrates to a deep position inside the substrate, so that it reaches the long wavelength detection region and generates a photocharge. On the other hand, light having a relatively short wavelength component generates photoelectric charge in a short wavelength detection region at a shallow position inside the substrate. The electric signals derived from the photocharges generated in these different regions are individually extracted by the signal extracting means.

  Conventionally, when fluorescence detection is performed by one image sensor (or camera), for example, it has been necessary to detect two-wavelength fluorescence in a time-sharing manner, but by using the biological tissue measurement image sensor according to the third invention. Two wavelengths of fluorescence can be detected at the same time, not in time division, that is, under exactly the same conditions in terms of time. Conventionally, in order to avoid time lag, an image is divided into two by an optical system in a microscope and fluorescence detection is performed by two image sensors (or cameras). In some cases, there is no time shift, but a spatial shift depending on the optical system is unavoidable. On the other hand, by using the biological tissue measurement image sensor according to the third aspect of the invention, it is possible to detect the fluorescence light intensity at exactly the same location without any spatial deviation as described above. For this reason, for example, when calculating the light intensity ratio of two-wavelength fluorescence, the light intensity under the same conditions can be compared both in terms of time and space, so that the measurement accuracy is improved.

A biological tissue measurement image sensor according to a fourth aspect of the invention made to solve the above-described problem is a biological tissue measurement image sensor for acquiring a two-dimensional image based on a light phenomenon of a biological tissue that is an observation object. Because
a) One minute light receiving element, two first and second charge accumulating units, and for selectively transferring charges from the one minute light receiving element to the first and second charge accumulating units. One pixel cell is configured to include a charge transfer unit and two first and second output units that output voltages according to the accumulated charges of the first and second charge accumulation units, A photoelectric conversion unit in which pixel cells are two-dimensionally arranged;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
And irradiating the observation object with two types of excitation light having different wavelengths from each other in a time-sharing manner in a state where the observation object is in contact with the protective film layer, and depending on the excitation light, the observation object The charge signals generated in each micro light receiving element by the light emitted from the object are distributed by the charge transfer unit in synchronization with the time division switching timing of the two types of excitation light and accumulated in the first and second charge storage units. Thereafter, the output is performed from the first and second output units, respectively.

  The biological tissue measurement image sensor according to the fourth invention is suitable for, for example, two-wavelength excitation / one-wavelength fluorescence measurement. In the two-wavelength excitation / one-wavelength fluorescence measurement, the living tissue disposed so as to be in contact with the protective film layer is alternately irradiated with excitation light having two different wavelengths in a time division manner. The fluorescence generated in the living tissue by each excitation light enters the same minute light receiving element and is photoelectrically converted to generate electric charge, but it is driven by the charge transfer unit driven in synchronization with the time division switching timing of the excitation light. The charge is distributed for each charge derived from the fluorescence corresponding to each excitation light, and is accumulated in each charge accumulation section. Therefore, if the biological tissue measurement image sensor according to the fourth aspect of the invention is used, two systems of signals derived from fluorescence with respect to two-wavelength excitation can be extracted at the same time. For example, processing such as comparing the light intensities of the two is easy. Yes.

  Furthermore, in the excitation light irradiation / fluorescence detection measurement in various forms as described above, the fluorescence light intensity is generally much smaller than the excitation light intensity. For this reason, even if the signal conversion means is introduced as in the second invention, for example, it may not be possible to obtain sufficient detection sensitivity and accuracy. Therefore, in such a case, it is effective to relatively reduce the influence of the excitation light.

A biological tissue measurement image sensor according to the fifth invention for the above purpose is a biological tissue measurement image sensor for obtaining a two-dimensional image by a light phenomenon of a biological tissue that is an observation target,
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) While blocking the passage of the wavelength band of the excitation light irradiated to the observation object arranged in contact with the protective film layer, the wavelength of the fluorescent light emitted from the observation object in response to the excitation light An optical filter layer that passes the band;
It is characterized by having.

  Here, the optical filter layer can be an absorption filter that selectively absorbs light in the wavelength band of excitation light or an interference filter that selectively reflects light in the wavelength band of excitation light. Further, when the imaging element included in the sensor has a CMOS structure, since a polycrystalline silicon layer is used for a gate or the like, it can be used as an optical filter layer for blocking excitation light.

  According to the biological tissue measurement image sensor according to the fifth aspect of the present invention, the light intensity of the excitation light incident on the minute light receiving element is significantly reduced by the optical filter layer, so that the light intensity of the fluorescence is relatively increased. Increased fluorescence detection.

  In addition, in order to achieve the above object, by utilizing the characteristic of the sensitivity curve that the detection sensitivity is relatively lowered due to light absorption near the surface inside the substrate on the short wavelength side in the silicon solid-state imaging device. It is conceivable to remove photocharges generated by excitation light that is visible light on the ultraviolet light or short wavelength side.

That is, the image sensor for biological tissue measurement according to the sixth invention is an image sensor for biological tissue measurement for acquiring a two-dimensional image due to a light phenomenon of the biological tissue that is an observation object,
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) To discharge charges generated near the substrate surface of the micro light-receiving element as unnecessary charges by the relatively short-wavelength excitation light applied to the observation object placed in contact with the protective film layer. Charge discharging means,
d) a signal extracting means for extracting the charges generated in the deep part of the substrate of the micro light receiving element by the fluorescence emitted from the observation object in response to the excitation light as effective charges;
It is characterized by having.

  Here, for example, a photogate or the like can be used as the charge discharging means. In this configuration, the excitation light having a relatively short wavelength generates photocharges near the substrate surface directly under the photogate, but these charges are discharged by the charge discharging means and do not contribute to the output. On the other hand, the fluorescence having a longer wavelength than the excitation light penetrates deep into the substrate to generate a photocharge, and a signal due to this charge is taken out by the signal takeout means and used for image formation and the like. Therefore, by extracting only the electric signal derived from the photocharge generated in the deep part of the substrate in this way, a signal with reduced influence of the excitation light can be obtained.

  Also with the image sensor for biological tissue measurement according to the sixth aspect of the invention, the light intensity of the excitation light incident on the micro light receiving element is significantly reduced, as in the case of the fifth aspect of the invention. Thus, the detection of fluorescence is enhanced.

  As a biological tissue measuring method using the biological tissue measuring image sensor according to the first to sixth inventions, roughly two methods can be considered. One of them is that the living tissue isolated from the living body or cultured in vitro is directly mounted on the protective film layer of the image sensor for measuring living tissue according to any one of the above-described inventions. This is a method for acquiring a two-dimensional image. In this measurement method, various types of fluorescence detection that can be observed under a conventional microscope can be performed.

  On the other hand, in another measurement method, the biological tissue measurement image sensor according to any one of the above inventions is implanted in a living organism, and the protective film layer is directly contacted with the biological tissue inside the organism. This is a method of acquiring a two-dimensional image due to the light phenomenon of the living tissue in a state of being in contact. In this method, although there is a limitation on the fluorescent label of the biological tissue to be observed, the biological information can be observed in a normal activity state, and biological information that has conventionally been difficult to obtain is relatively Can be easily obtained.

[First embodiment]
One embodiment of a biological tissue measurement image sensor according to the present invention and a biological tissue measurement method using the same will be described with reference to the drawings.
FIG. 1 is a plan view showing a schematic structure of an image sensor 1 used in the sensor unit 2 according to the first embodiment, and FIG. 2 is a schematic longitudinal section of the sensor unit 2. The image sensor 1 is a photoelectric conversion region 11 in which a large number of minute light receiving elements are two-dimensionally arranged on a semiconductor substrate 10 such as silicon, and a signal for reading signals from each pixel cell included in the photoelectric conversion region 11. A column selection control circuit 17 and a row selection control circuit 18 are provided.

  A total of 64 pixel units 12 of 8 columns vertically and 8 rows horizontally are provided in the photoelectric conversion region 11, and each pixel unit 12 has a total of 225 pixels of 15 columns vertically and 15 rows horizontally. An area exists. However, among the 225 pixel regions in each pixel unit 12, the central 25 pixel regions are not used for light reception but are used for the protruding electrode regions. That is, a substantially square electrode forming pad 15 is provided in the 25 pixel regions, and a metal protruding electrode 16 protruding upward in a substantially cylindrical shape is formed thereon. In each pixel unit 12, 200 pixel regions surrounding the central electrode forming pad 15 are allocated to the light receiving pixel cells 13, respectively. Each pixel cell 13 includes a photodiode 14 as a micro light receiving element in the present invention, and various circuits for converting a current signal obtained by the photodiode 14 into a voltage signal and amplifying the voltage signal. Including.

  Therefore, in this imaging device 1, a total of 64 protruding electrodes 16 are provided in the photoelectric conversion region 11 at substantially equal intervals in the vertical direction and the horizontal direction, and 12800 pixel cells 13 are provided. The size of one pixel region, the size of the electrode forming pad 15 and the like can be determined as appropriate according to the semiconductor process used for manufacturing, the purpose of this sensor, and the like. The pad can be 25 μm square. Of course, the size and the number of pixels are not limited to those described here. In addition, although it is comprised so that the electrical signal acquired by photoelectric conversion for every pixel cell 13 can be taken out serially like the conventional solid-state image sensor, it is comprised with respect to the protruding electrode 16, respectively. It is configured such that signal extraction and current injection can be performed independently.

  In the sensor unit 2 of the present embodiment, as shown in FIG. 2, a wire bonding pad formed on the image sensor 1 by fixing the image sensor 1 having the above configuration onto a sensor substrate 20 made of, for example, polyimide resin. And an electrode portion provided on the sensor substrate 20 are electrically connected by a wire 23 such as aluminum or gold. Further, the entire surface of the image sensor 1 and the wire 23 are covered with a protective layer 22 made of epoxy resin or the like. However, only the upper end portion of the protruding electrode 16 is exposed on the protective layer 22. Since this sensor unit 2 is used in a state in which it is in direct contact with a living tissue and, in some cases, kept in that state for a considerably long period of time, it protects circuits, wirings, and the like from erosion by biological fluids including electrolytes. There is a need. On the other hand, since the protective layer 22 itself is in direct contact with the living tissue, it is also necessary to avoid that the protective layer 22 itself damages the living tissue or inhibits physiological activity. For this reason, the protective layer 22 is made of a material that does not harm the living tissue such as an epoxy resin.

  FIG. 3 is a diagram showing a circuit configuration in one pixel cell 13, and FIG. 4 is a waveform diagram for explaining the operation of this circuit. Here, a normal CMOS active pixel sensor that outputs an analog voltage value according to the amount of incident light so that extremely weak light emission or fluorescence from a living cell or the like can be detected (see FIG. 4A). In addition, a circuit for enabling operations of a pulse frequency modulation (PFM) system and a pulse width modulation (PWM) system is incorporated.

  In other words, the pixel cell 13 photoelectrically converts incident light into an electrical signal, and also functions as a charge storage unit that stores charges generated by photoelectric conversion by the junction capacitance, and erases the stored charges. A reset gate switch unit 32 for amplifying, amplifying units 33 and 34 for amplifying electric charges and outputting them as voltage signals, a comparator 35 for comparing the amplified voltage signals with a predetermined reference voltage Vref, and a comparator output A flip-flop circuit 36 that reads as a data input by a clock signal CLK of a predetermined frequency, an AND gate 37 that receives a flip-flop output and a reset timing signal and sends a reset signal to the gate switch unit 32, and an inverter 38 that inverts the output are included. .

  First, the operation when the above configuration is operated by the PFM method will be described. When the reset signal RST is input and the gate switch unit 32 is turned on, the potential VPD of the photodiode 31 is charged to the vicinity of VDD, and when the gate switch unit 32 is turned off, the potential VPD of the floating photodiode 31 is discharged by photocharge. Decreases with quantity. Therefore, the lowering of the potential VPD becomes faster as the incident light intensity on the photodiode 31 is higher. When the potential VPD ′ corresponding to the potential VPD falls below Vref, the output of the comparator 35 changes from L → H. In response to this, the output of the flip-flop circuit 36 changes from L to H when the clock signal CLK rises next time. Then, with an appropriate delay from this change, the output of the AND gate 37 gives the rising reset signal RST to the gate switch section 32, so that the potential VPD of the photodiode 31 rises again to the vicinity of VDD. As a result, the output of the comparator 35 changes from H → L. Therefore, when the clock signal CLK rises next time, the output of the flip-flop circuit 36 changes from H → L.

  The width of the output pulse of the flip-flop circuit 36 is always constant because it depends on the delay time of the AND gate 37 and is independent of the incident light intensity. However, the frequency of generation of the output pulse is fast at the decreasing rate of the potential VPD. As the incident light quantity increases, the pulse frequency increases (see FIG. 4B). The circuit that has received this output pulse integrates the number of pulses, but the integration of the number of pulses when a large number of measurements are performed has the same effect as taking the average of the many measurements. Therefore, this PFM method has a feature that a change in incident light intensity can be detected with high accuracy. Therefore, for example, it is particularly suitable when it is necessary to catch a minute change in light intensity due to fluorescence under the condition that strong excitation light is simultaneously present, such as observation of a fluorescent protein.

  Next, the operation when the above configuration is operated by the PWM method will be described. Although the basic operation is the same as that of the PFM method, in the PWM method, when the reset signal RST is input and the gate switch unit 32 is turned on, the potential VPD of the photodiode 31 is charged to the vicinity of VDD, thereby the comparator 35. From the time when the output of H changes from H to L, the potential VPD decreases according to the amount of discharge due to photocharge, and the time span from when the potential VPD 'falls below Vref and the output of the comparator 35 changes from L to H The pulse signal is obtained. The smaller the amount of incident light, the slower the potential VPD decreases, so the pulse width becomes longer (see FIG. 4C). Therefore, this PWM method has a feature that even if very weak light is incident, a slight difference in the weak light intensity is detected as being enlarged as a difference in pulse width. Therefore, it is particularly suitable when the incident light intensity is extremely weak, such as when observing photoproteins.

  As described above, in this imaging device, one of the PFM method and the PWM method is appropriately selected according to the purpose, and a pulse signal sequence corresponding to the incident light intensity can be extracted as an output.

Next, a specific example of a biological tissue measurement method using the sensor unit 2 having the above-described configuration will be described.
One of the typical measurement methods is in vitro. That is, as shown in FIG. 5, light emission / fluorescence imaging is performed in a state where an isolated cell collected from a living body or a cell S cultured in a test tube or the like is directly placed on the protective layer 22 of the sensor. At this time, since a part of the protruding electrode 16 contacts the cell S to be observed, by selectively applying a predetermined potential to the protruding electrode 16, the cell S is locally applied to the portion in contact with the cell S. Can be applied with electrical stimulation. In addition, information by an electrical signal inside the cell S may be acquired by detecting a voltage generated in the protruding electrode 16 instead of applying a voltage. Such electrical stimulation or electrical measurement and imaging by light emission / fluorescence imaging can be appropriately combined.

  Another representative measurement method is in vivo. That is, the sensor is implanted in the living tissue of a laboratory animal such as a mouse in a living state, and a signal acquired by the sensor is transmitted to an external device by wire or wireless to obtain an image by imaging. In such measurement, it is possible to observe a living tissue while maintaining a normal activity state. Of course, such a measurement can be combined with electrical stimulation or electrical measurement in the same manner as in vitro.

[Second Embodiment]
Next, an image sensor according to a second embodiment will be described with reference to FIG. FIG. 6 is a schematic longitudinal sectional view of one pixel cell 13 of the image sensor according to the second embodiment.
Fluorescence measurement often used in current bioimaging technology irradiates an observation object with excitation light of a predetermined wavelength, and creates a fluorescence image by measuring excitation light generated by exciting a fluorescent dye or the like by the excitation light. . In such a measurement, excitation light as well as fluorescence enters the photoelectric conversion area of the image sensor at the same time. Therefore, in order to measure the intensity change of fluorescent light, which is the original measurement target, with high accuracy, the influence of excitation light is removed as much as possible. It is important to.

  In general, since excitation light needs to be given high energy, visible light on the short wavelength side is used even for ultraviolet light or visible light. On the other hand, the fluorescence emitted from the fluorescent dye or the like has a longer wavelength than the excitation light. Therefore, in the image sensor according to the second embodiment, as shown in FIG. 6, the excitation light wavelength band is blocked on the upper surface of the photodiode 14 of each pixel cell 13, and the characteristic is such that the fluorescence wavelength band is allowed to pass. An optical filter layer 41 is provided. Specifically, as the optical filter layer 41, after forming a pixel circuit by a normal semiconductor manufacturing process, an absorption filter layer having a characteristic of absorbing the wavelength band of excitation light is formed on the protective film, or What is necessary is just to form the interference film filter layer which has the characteristic which reflects the wavelength range | band of excitation light.

  In a CCD or CMOS process, a polycrystalline silicon layer is used to form a gate electrode and the like. In general, the standard film thickness of such a polycrystalline silicon layer is about 300 nm, but even with such a film thickness, it absorbs most of ultraviolet light and short wavelength visible light used as excitation light. Is a sufficient film thickness. Therefore, for example, by adopting a photogate structure for the photodiode 14, the polycrystalline silicon layer 42 formed for this gate structure can be used as an absorption filter for excitation light (see FIG. 7).

[Third embodiment]
The purpose of the image sensor according to the third embodiment is the same as that of the second embodiment. However, the charge generated by being incident on the photodiode 14 rather than blocking the excitation light before reaching the photodiode 14. Of these, those derived from excitation light are removed. That is, considering the wavelength dependence of the absorption coefficient of silicon, which is the material of the semiconductor substrate, the longer the wavelength of light, the deeper the substrate. As described above, normally, the excitation light has a short wavelength and the fluorescence has a long wavelength. Therefore, when excitation light and fluorescence enter the photodiode at the same time, the fluorescence reaches a deeper part of the substrate and generates a charge in the vicinity thereof, whereas the excitation light does not reach the deep part of the substrate and mainly the substrate. An electric charge is generated near the surface.

  Therefore, in the image sensor according to the third embodiment, as shown in FIG. 8, a PN junction for detecting fluorescence is formed in a relatively deep portion of the substrate 10, and charge discharge by the photogate 43 is performed on the surface of the substrate 10. Means are provided. The fluorescence having a relatively long wavelength reaches the deep part of the substrate 10 and generates a photocharge in the vicinity of the long wavelength detection region P2, but the excitation light having a relatively short wavelength is a short wavelength located in a shallow part immediately below the photogate 43. Photocharge is generated in the vicinity of the detection region P1. By discharging the charges accumulated in the short wavelength detection region P1 from the electrode 44, it is possible to detect only the photocharge derived from the fluorescence in the photodiode 14 and to detect it. Note that a PN junction may be used instead of the photogate structure.

  In this structure, the depth of the depletion layer immediately below the photogate (or PN junction) can be changed by controlling the bias voltage applied to the photogate (or PN junction) provided on the substrate surface. Therefore, it is possible to adjust the operating conditions such as how far the generated charges are discharged, and the operating conditions are determined appropriately in consideration of the wavelength difference between excitation light and fluorescence. Just do it. When the photogate structure is used, as described in the second embodiment, a filter effect of absorbing short wavelength excitation light by the polycrystalline silicon layer of the photogate can be expected.

[Fourth embodiment]
Next, an image sensor according to a fourth embodiment will be described with reference to FIGS. As one method of the above-described fluorescence measurement, an observation object is irradiated with excitation light having a predetermined wavelength, and fluorescence emitted by the fluorescent dye is detected at two different wavelengths when the excitation light is irradiated. There is a technique called one-wavelength excitation and two-wavelength detection ratiometry. For example, FIG. 9 shows an emission spectrum at the time of labeling with a fluorescent dye Indo-1 having calcium Ca reactivity. Thus, since the fluorescence spectrum is different between the case where Ca + is present and the case where Ca + is not present, the presence of Ca + is obtained by detecting the light intensity of the fluorescence simultaneously at the peak wavelengths of both spectra and determining the light intensity ratio. It can be determined whether or not.

  The image sensor of the fourth embodiment is for detecting such two-wavelength fluorescence. Basically, as in the third embodiment, the wavelength dependency of the degree of penetration of incident light in the substrate depth direction is shown. Use. That is, in the CMOS manufacturing process, a structure in which a plurality of PN junctions are arranged in the depth direction of the substrate at the same position in the substrate can be formed. For example, when the P-diff is present in the N-Well structure, there are two PN junctions between the N-Well and the P-type substrate and between the P-diff and the N-Well. Since the absorption coefficient of silicon differs depending on the wavelength of incident light, the signal intensity at the plurality of PN junctions depends on the wavelength. Therefore, in the imaging sensor according to the fourth embodiment, as shown in FIG. 10, in the photodiode 14, the deep PN junction is the long wavelength detection region Q1, and the shallow PN junction is the short wavelength detection region Q2, respectively. The signal readout circuits 51 and 52 are provided independently. Thereby, when the fluorescence from a biological tissue is detected by the measurement method as shown in FIG. 5 using this image sensor, two different wavelengths of fluorescence can be obtained simultaneously.

  The fluorescence obtained at this time has not only a temporal shift but also a spatial shift. Therefore, if biological tissue measurement is performed using the image sensor according to the fourth embodiment, extremely high accuracy can be obtained when calculating the intensity ratio of two-wavelength fluorescence.

[Fifth embodiment]
Next, an image sensor according to a fifth embodiment will be described with reference to FIGS. As another method of fluorescence measurement, two different wavelengths of excitation light are alternately irradiated in time division on the observation object, the emission state when each excitation light is irradiated is measured, and the emission intensity at the same location is measured. There is a technique called a two-wavelength excitation / one-wavelength detection ratiometry to be compared. For example, pH-sensitive GFP can be estimated by introducing pH-sensitive GFP into a living tissue and examining the intensity ratio of two fluorescences by two-wavelength excitation (410 nm, 488 nm).

  The image sensor of the fifth embodiment is for detecting two fluorescences simultaneously in such time division excitation of two wavelengths. As shown in FIG. 11, one pixel cell 60 includes a first photogate (PG) 61 and first and second charge accumulating units for accumulating photoelectric charges generated in the photogate 61. The second floating diffusion layers (FD) 62 and 63 and the first charge transfer unit for transferring the signal charges accumulated in the photogate 61 to the first and second floating diffusion layers 62 and 63 at a predetermined timing. 1 and second transfer gates (TG) 64 and 65, and output units 66 and 67 for amplifying signal charges accumulated in the first and second floating diffusion layers 62 and 63 and outputting them as voltages.

  When performing two-wavelength excitation and one-wavelength detection, for example, as shown in FIG. 5, excitation light having two predetermined wavelengths λ1 and λ2 is applied to a living tissue placed on the image sensor at a predetermined frequency. Irradiate in time division. In response to this, two fluorescent lights corresponding to the two-wavelength excitation are emitted from the living tissue and enter the photogate 61 of each pixel cell 60. In the pixel cell 60 configured as described above, the circuit is operated in synchronization with the excitation light switching cycle. That is, during the irradiation period of the excitation light having the first wavelength λ 1, fluorescence corresponding to the excitation light having the first wavelength λ 1 is incident on the photogate 61, and signal charges corresponding to the received light intensity are generated immediately below the photogate 61. (FIG. 12A). Immediately before this period ends, the first transfer gate 64 is opened, and signal charges are transferred and accumulated in the first floating diffusion layer 62 (FIG. 12B). On the other hand, in the irradiation period of the excitation light having the second wavelength λ2, fluorescence corresponding to the excitation light having the second wavelength λ2 is incident on the photogate 61, and signal charges corresponding to the received light intensity are generated immediately below the photogate 61. (FIG. 12A). Then, immediately before the end of this period, the second transfer gate 65 is opened, and the signal charge is transferred and accumulated in the second floating diffusion layer 63 (FIG. 12C).

  By repeating the above operation many times during the period of one frame in synchronization with the switching period of the excitation light, the signal charge corresponding to the fluorescence derived from the excitation light having the first wavelength λ 1 is stored in the first floating diffusion layer 62. In the second floating diffusion layer 63, signal charges corresponding to fluorescence derived from the excitation light having the second wavelength λ2 are added and accumulated. Then, after the end of one frame period, the respective accumulated accumulated charges are taken out as voltages through the first and second output sections 66 and 67 (FIG. 12D). As a result, signals constituting two fluorescent images can be obtained simultaneously.

When the pixel cell 60 is formed by a general CMOS process, parasitic n ⁺ diffusion between the photogate 61 and the first and second transfer gates 64 and 65 as illustrated in FIG. Layer 68 is formed. Since the parasitic diffusion layer 68 forms a potential pocket between the photogate 61 and the transfer gates 64 and 65, signal charges are transferred from the photogate 61 to the floating diffusion layers 62 and 63 via the transfer gates 64 and 65. In this case, some signal charges are captured. Therefore, in order to reduce such influence, a MOS transistor 69 is provided in which the parasitic diffusion layer 68 is a drain (or source) and the source (or drain) is connected to the reset power supply line Vrs.

  Then, signal charges are accumulated in the depletion layer formed immediately below the photogate 61, and the MOS transistor 69 is turned on when the potential immediately below the photogate 61 rises as shown in FIG. The potential of the diffusion layer 68 is set to a value close to Vrs. That is, by raising the potential of the parasitic diffusion layer 68 prior to the transfer of the signal charge, the bottom of the potential pocket formed in that portion is made shallower. As a result, when the transfer gate 64 or 65 is opened, the signal charge is not trapped in the potential pocket or even if it is trapped, the influence of the parasitic diffusion layer 68 can be reduced.

  In this way, if biological tissue measurement is performed using the image sensor according to the fourth embodiment, signals corresponding to two fluorescences for two-wavelength excitation can be obtained simultaneously, and the process for calculating the intensity ratio is also simple. become.

  It should be noted that each of the above embodiments is an example, and it is obvious that even if appropriate changes and modifications are made within the scope of the present invention, they are included in the scope of the claims of the present application.

1 is a plan view showing a schematic structure of an image sensor used in a sensor unit according to an embodiment (first embodiment) of the present invention. The schematic longitudinal cross-section of the sensor unit by 1st Example. FIG. 3 is a circuit configuration diagram of one pixel cell of the image sensor in the first embodiment. FIG. 4 is a waveform diagram for explaining the operation of the circuit of FIG. 3. Schematic when the sensor unit according to the first embodiment is applied to in vitro measurement. The schematic longitudinal cross-sectional view of one pixel cell of the image sensor by 2nd Example. The schematic longitudinal cross-sectional view of one pixel cell of the image sensor by the modification of 2nd Example. FIG. 10 is a schematic longitudinal sectional view of one pixel cell of an image sensor according to a third embodiment. The figure which shows the emission spectrum in the case of labeling with fluorescent dye Indo-1. The schematic block diagram of the light-receiving part of the image sensor by 4th Example. FIG. 10 is a schematic configuration diagram of one pixel cell of an image sensor according to a fifth embodiment. Explanatory drawing of operation | movement of the pixel circuit in 5th Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Imaging device 10 ... Semiconductor substrate 11 ... Photoelectric conversion area | region 12 ... Pixel unit 13 ... Pixel cell 14, 31 ... Photodiode 15 ... Electrode formation pad 16 ... Projection electrode 17 ... Column selection control circuit 18 ... Row selection control circuit 20 ... Sensor substrate 22 ... Protective layer 23 ... Wire 32 ... Gate switch part 33 ... Amplifying part 35 ... Comparator 36 ... Flip-flop circuit 37 ... AND gate 38 ... Inverter 41 ... Optical filter layer 42 ... Polycrystalline silicon layer 43 ... Photo gate 44 ... Electrodes 51, 52 ... Signal readout circuit

Claims (10)

An image sensor for measuring a living tissue for acquiring a two-dimensional image due to a light phenomenon of a living tissue that is an observation object,
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) In the region of the photoelectric conversion unit, the upper end of the photoelectric conversion unit is exposed or protruded from the protective film layer, and is used to apply electrical stimulation to the observation object and / or to localize the observation object. An electrode part for detecting a potential,
An image sensor for measuring a biological tissue, wherein the observation object is imaged in a state where the observation object is in contact with the protective film layer.   2. The biological tissue measurement image sensor according to claim 1, wherein a plurality of the electrode portions are two-dimensionally arranged at predetermined intervals in the region of the photoelectric conversion portion. An image sensor for measuring a living tissue for acquiring a two-dimensional image due to a light phenomenon of a living tissue that is an observation object,
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) a signal conversion means for converting the light intensity signal received by each of the micro light receiving elements into a pulse signal sequence having information on the pulse width or pulse frequency by pulse width modulation or pulse frequency modulation;
An image sensor for measuring a living tissue, wherein the observation object is imaged in a state where the observation object is in contact with the protective film layer. An image sensor for measuring a living tissue for acquiring a two-dimensional image due to a light phenomenon of a living tissue that is an observation object,
a) A large number of micro light receiving elements are two-dimensionally arranged, and each micro light receiving element is formed in a shallow portion inside the substrate and mainly includes a short wavelength detection region for detecting light having a relatively short wavelength, A photoelectric conversion unit having a long wavelength detection region that mainly detects light having a relatively long wavelength formed in a deeper portion inside the substrate than the short wavelength detection region;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) a signal extraction means for independently extracting an electric signal generated by photoelectric conversion in the short wavelength detection region and an electric signal generated by photoelectric conversion in the long wavelength detection region;
In the state where the observation object is in contact with the protective film layer, an electrical signal for light emitted from the observation object at two different wavelengths is transmitted in the short wavelength detection region and the long wavelength detection region. An image sensor for measuring a living tissue, which is obtained simultaneously. An image sensor for measuring a living tissue for acquiring a two-dimensional image due to a light phenomenon of a living tissue that is an observation object,
a) One minute light receiving element, two first and second charge accumulating units, and for selectively transferring charges from the one minute light receiving element to the first and second charge accumulating units. One pixel cell is configured to include a charge transfer unit and two first and second output units that output voltages according to the accumulated charges of the first and second charge accumulation units, A photoelectric conversion unit in which pixel cells are two-dimensionally arranged;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
And irradiating the observation object with two types of excitation light having different wavelengths from each other in a time-sharing manner in a state where the observation object is in contact with the protective film layer, and depending on the excitation light, the observation object The charge signals generated in each micro light receiving element by the light emitted from the object are distributed by the charge transfer unit in synchronization with the time division switching timing of the two types of excitation light, and are stored in the first and second charge storage units. Then, an image sensor for measuring a living tissue, which is output from the first and second output units, respectively. An image sensor for measuring a living tissue for acquiring a two-dimensional image due to a light phenomenon of a living tissue that is an observation object,
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) While blocking the passage of the wavelength band of the excitation light irradiated to the observation object arranged in contact with the protective film layer, the wavelength of the fluorescent light emitted from the observation object in response to the excitation light An optical filter layer that passes the band;
An image sensor for measuring a living tissue, comprising:   7. The image sensor for biological tissue measurement according to claim 6, wherein the image sensor included in the sensor has a CMOS structure, and a polycrystalline silicon layer for forming the CMOS structure is used as the optical filter layer. An image sensor for measuring biological tissue. An image sensor for measuring a living tissue for acquiring a two-dimensional image due to a light phenomenon of a living tissue that is an observation object,
a) a photoelectric conversion unit in which a large number of minute light receiving elements are arranged two-dimensionally;
b) a protective film layer made of a material that does not harm living tissue, covering the photoelectric conversion part;
c) To discharge charges generated near the substrate surface of the micro light-receiving element as unnecessary charges by the relatively short-wavelength excitation light applied to the observation object placed in contact with the protective film layer. Charge discharging means,
d) a signal extracting means for extracting the charges generated in the deep part of the substrate of the micro light receiving element by the fluorescence emitted from the observation object in response to the excitation light as effective charges;
An image sensor for measuring a living tissue, comprising:   A biological tissue isolated from a living body or cultured outside a living body in a two-dimensional state due to a light phenomenon of the living tissue in a state where the living tissue is directly placed on the protective film layer of the image sensor for biological tissue measurement according to claim 1. A biological tissue measurement method characterized by acquiring an image.   The image sensor for measuring biological tissue according to claim 1 is embedded in a living body, and the two-dimensional due to a light phenomenon of the living tissue while the protective film layer is in direct contact with the living tissue inside the living body. A biological tissue measurement method characterized by acquiring an image.

Images