JP2932228B2 - Densitometer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a densitometer for measuring the distribution of absorbance of a sample, and more particularly to a method for measuring reflected light.
This type of densitometer has high measurement sensitivity,
The present invention relates to a densitometer capable of measuring the absorbance of a substance to be measured distributed two-dimensionally.

[0002]

2. Description of the Related Art Conventionally, a densitometer for measuring the concentration of a sample spot spread on a support such as an electrophoresis gel has been disclosed in Japanese Patent Application Laid-Open No. 56-46448. Scan in the spot development direction, measure the transmitted light, and at each scan stroke,
The concentration of the sample or the substance is measured by performing relative measurement of the peak value based on the difference in absorbance between the portion having the sample spot and the portion of the support alone.

[0003]

However, in a densitometer based on such a method of measuring transmitted light, the absorbance of the sample or the substance spread on a highly reflective support such as a nylon film or a nitrocellulose film is used. concentration)
Cannot make accurate measurements.

In a densitometer for measuring absorbance (density) using reflected light, the background optical axis becomes large because the optical axis on the light receiving side is the main axis of the light reflected by the sample surface. Therefore, when reading a concentration distribution pattern of a fluorescent substance, which measures a fluorescence signal by labeling a sample with a fluorescent dye or the like, there is a problem that noise is large and measurement sensitivity is difficult to increase. Since the measurement sensitivity cannot be increased in this way,
There is also a problem that weak fluorescence cannot be measured.

An object of the present invention is to solve such a problem. An object of the present invention is to provide a densitometer for measuring the distribution of absorbance of a sample, which has a high measurement sensitivity and a two-dimensional distribution. An object of the present invention is to provide a densitometer capable of measuring the absorbance of a substance to be measured.

Another object of the present invention is to provide a densitometer which can measure the absorbance of a substance to be measured distributed two-dimensionally and can be used for reading a distribution pattern of a fluorescent substance.

[0007]

In order to achieve the above-mentioned object, a densitometer according to the first aspect of the present invention comprises a sample support adsorbing or attaching to a sample support having light scattering properties.
The sample is dyed or labeled as it is or using a specific substance on the sample, and the irradiation light is applied to the sample support.
And irradiate the sample with the sample support and the sample from the sample support.
Shako measured, a <br/> den citrate meter measuring the absorbance of the sample or substance, a light source for emitting the illumination light, the thickness direction of the waving optical axis of irradiation light the sample support Irradiation light
A light scanning mechanism that scans the light, sets a light receiving surface in a direction different from the optical axis of the irradiation light, and irradiates the sample support with irradiation light
Arranged on the side of irradiating a light receiving unit for selectively receiving scattered light from the sample support and sample by the spatial positional relationship between the light-receiving path, and photoelectrically converts an optical signal received by the light receiving portion, the electrical A photoelectric conversion unit that outputs a signal, a data acquisition unit that acquires data corresponding to scanning of irradiation light by an optical scanning mechanism with respect to the electric signal from the photoelectric conversion unit, and calculates the absorbance of the sample based on the acquired data. A data processing unit is provided.

Further, a densitometer according to a second feature of the present invention is a method in which a sample is adsorbed on a sample support having light scattering properties.
Others deposited, staining or labeling the sample as such or with a specific substance in the sample, the illumination light sample
Scanning and irradiating the support, reflected light from the sample support
To measure the absorbance of the sample or the substance.
A citrate meter, a light source for emitting the illumination light, the irradiation light
Irradiating light in the thickness direction of the sample support by shaking the optical axis of
An optical scanning mechanism to be inspected, a light receiving surface set in a direction different from the optical axis of the irradiation light, and irradiation of the sample support with the irradiation light.
Arranged on the side of morphism, selectively receiving scattered light from the intersection optical axis of the light receiving portion in the sample surface with respect to the optical axis of the irradiation light, a sample support by the spatial positional relationship between the light receiving path and the sample a light receiving unit for photoelectrically converts the optical signal received by the light receiving unit, and a photoelectric conversion unit that outputs an electric signal, and amplified in correspondence with the scanning of the irradiation light to the electric signal from the photoelectric conversion unit, an electric A data acquisition unit for acquiring data from a signal and a data processing unit for calculating the absorbance of a sample based on the acquired data are provided.

In the densitometer according to a third aspect of the present invention, the light source is a laser, and the substance for staining or labeling the sample is Coomassie brilliant blue or an analog thereof, amide black or silver stain, or gold colloid. It is characterized in that at least one of the dyeings is used.

[0010]

Further, in a densitometer according to a fourth aspect of the present invention, the densitometer adsorbs to a sample support having light scattering properties.
Alternatively, the fluorescent substance labeled on the attached sample
Excitation and emission of fluorescent light
These scattered lights are measured, and the absorbance distribution is measured.
And reading a fluorescent pattern that emits light .

[0012]

In the densitometer having such a feature, when the light source emits irradiation light, the light scanning mechanism swings the optical axis of the irradiation light from the light source to attach the sample to the sample support.
The irradiation light is scanned in the thickness direction of the body . The light-receiving unit, sample Hoyo
In order to measure the reflected light from the sample support and the sample support, set the light receiving surface in a direction different from the optical axis of the irradiation light , and
It is arranged on the side that irradiates the body with irradiation light, and selectively receives scattered light from the sample and the sample support according to the spatial positional relationship with the light receiving path. The photoelectric conversion unit photoelectrically converts an optical signal received by the light receiving unit and outputs an electric signal.
When the electric signal is output, the data acquisition unit acquires data corresponding to the scanning of the irradiation light with respect to the electric signal from the photoelectric conversion unit. Then, the data processing unit calculates the absorbance of the sample based on the acquired data. In this way, the light scattering
Adsorbing or adhering the sample to a sample support having disturbance,
The sample is stained or labeled as it is or with a specific substance, and the irradiated light is scanned on the sample support.
And irradiate the sample to measure the reflected light (scattered light) from the sample support.
And the absorbance of the sample or the substance is measured.

Further, in order to further increase the measurement sensitivity, the light receiving portion here is set with a light receiving surface in a direction different from the optical axis of the irradiation light, and is arranged closer to the light source than the sample surface. The optical axis intersects the optical axis of the irradiation light on the sample surface, and is configured to selectively receive scattered light from the sample and the sample support according to the spatial positional relationship of the light receiving path. Further, the data acquisition unit here is configured to amplify the electric signal from the photoelectric conversion unit in correspondence with the scanning of the irradiation light, and sequentially acquire data from the electric signal.

In this densitometer, as a light source
The substance that stains or labels the sample using a laser is Coomassie Britain.
liant Blue) or an analog thereof, Amido Black 10B or silver staining, or colloidal gold staining.
Thus, the absorbance of the sample can be measured with high sensitivity.

[0015]

Further, when a fluorescence pattern is measured using this densitometer, the sample is labeled with a fluorescent substance, the fluorescent substance is excited to emit fluorescence, and the emitted fluorescent pattern is compared with the absorbance of the sample. Read by measuring the distribution.

As described above, in the densitometer of the present invention, irradiation light for irradiating the sample is emitted from the light source, and the light emitted from the light source is scanned in the width direction of the measurement sample by the optical scanning mechanism. Further, by driving the sample stage in a direction perpendicular to the scanning direction of the irradiation light, two-dimensional measurement of the absorbance distribution becomes possible. In this case, by controlling the driving of the sample stage in steps smaller than the spot diameter of the irradiation light, it is possible to measure the distribution of the sample without omission.

Receiving the scattered light from the sample surface due to the irradiation light is performed by setting the light receiving surface corresponding to the width of the sample in one direction different from the optical axis direction.
By setting on the dimension, light is selectively received. The received light is condensed and taken into the photoelectric conversion unit, converted into an electric signal, and the converted electric signal is efficiently and efficiently amplified by an amplifier that performs an integrating operation corresponding to the scanning of the irradiation light. You.

When the absorbance of a sample is calculated from the data obtained by the data processing unit, when measuring the concentration of the sample or the substance, the measurement result of the scattered light of the sample or the substance whose concentration is known is As a relative measurement, the concentration of a sample or substance is measured. Regarding absorbance measurement, when the sample spot is on a thin film support such as a nitrocellulose membrane or nylon membrane used as a sample support having light scattering properties, when the sample spot is included for scattered light from the support alone Since the scattered light varies depending on the absorption of the sample, the relative absorbance is measured by the extinction coefficient specific to the substance. Even when the sample or the substance is stained with a specific substance, the absorbance of the sample or the substance is measured in the same manner.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be specifically described below with reference to the drawings. FIG. 1 is a block diagram showing a configuration of a main part of a measuring unit main body of a densitometer according to one embodiment of the present invention. In FIG. 1, 5 is a sample unit, 8 is a data processing device, 13 is scattered light, 21 is a light source, 22 is a vibrating mirror, 23 is a condenser, 24 is a photoelectric converter, 25 is an amplifier,
26 is an analog / digital conversion circuit, 27 is a control circuit, 28 is a storage circuit, 29 is an interface, 30 is a mirror driver, 31 is irradiation light such as a laser beam, 32
Is an optical trap for removing transmitted light, and 44 is a folding mirror.

First, an overall operation flow will be described with reference to FIG. Irradiation light 31 for irradiating the sample to be measured
Is emitted from the light source 21 and is irradiated on the turning mirror 44 while being scanned in the right and left directions in the figure by the vibrating mirror 22 driven by the mirror driver 30. Thus, the irradiation light 31 scanned by the vibration mirror 22 is reflected by the folding mirror 44 and then irradiated in the thickness direction while being scanned in the left and right directions in the drawing at the sample unit 5.

The scattered light 13 emitted from the sample section 5 by the irradiation of the spot light of the irradiation light 31 scanned by the vibration mirror 22 is received through the condenser 23. In the condenser 23, the optical axis of the light receiving path is spatially changed by the optical lens system so that the optical axis of the light receiving path for receiving the scattered light 13 is different from the optical axis of the spot light irradiating the sample unit 5. An optical path defining a positional relationship is formed.

As a result, the condenser 23 increases the sensitivity to the scattered light 13 emitted from the irradiation surface of the sample section 5 and collects the scattered light 13. Scattered light 13 condensed by condenser 23
Is further photoelectrically converted by a photoelectric conversion unit 24 including a condenser lens, an optical filter, and a photomultiplier,
An electric signal is output. The output electric signal is input to the amplifier 25.

The electric signal input to the amplifier 25 is further amplified by the amplifier 25 and input to an analog / digital converter (hereinafter, abbreviated as A / D converter) 26. The A / D converter 26 converts the input electric signal into digital data. The data of the detection signal corresponding to the scattered light 13 converted into digital data is stored in the memory 28, and the data stored in the memory 28 is sent to the data processing device 8 through the interface 29.
Then, data processing of signal processing is performed in the data processing device 8. The entire control of such a series of signal processing is performed by the control circuit 27.

Here, in order to measure the absorbance (concentration) of the sample, the sample (substance) to be measured supported by the support is irradiated with irradiation light, and the scattered light of the sample portion and the light of the support portion are irradiated. The scattered light is measured, and the concentration of the sample is measured by relative measurement based on the difference in scattered light between the samples on the support.
For this reason, measurement using various supports can be performed as a support for supporting the sample to be measured. The choice of the support is important because the concentration of the sample is measured by relative measurement based on the difference in scattered light between the samples on the support. For example,
When a white thin film filter such as a nylon membrane or a nitrocellulose membrane is used as a sample support, the transmittance (%) is about 10 ^-4 , and most of the light is scattered. Is performed.

As for the electrophoresis gel, for example,
In the polyacrylamide gel, the light is scattered only in the plane having a thickness of 0.35 mm, and the irradiation light is 80 to 9%.
0% is transmitted. The agarose gel is scattered in a plane direction within a plane having a thickness of about 5 mm, and thus has a transmittance of about 50%. As described above, for a support having a wide range of transmittance of 90% or less, the concentration of a sample can be measured with high measurement sensitivity by using a light-receiving system as described below.

Further, for a plurality of different samples,
Even when the same support such as a nitrocellulose membrane, a nylon membrane, and an electrophoresis gel is used, the absorbance of the sample to be measured is measured based on the difference in scattered light between the support and the sample. When the support is illuminated and when the sample is illuminated, the scattered light differs due to the absorption due to the specific extinction coefficient of the sample material. Can be measured as

Further, even when the sample is stained or labeled, the absorbance of the sample and the absorbance of the stained or labeled substance can be measured. As a substance to be stained or labeled with a sample, silver stain, colloidal gold stain, or amido black (Amido Black), and Coomassie Brilliant Blue (Coomassie Brilliant Blue) represented by the following chemical formula 1 At least one of the analogs is used.

Embedded image

Further, by adopting a configuration of a light receiving section and a photoelectric conversion section as described later, a fluorescent substance can be used for a sample labeled with a fluorescent substance by utilizing the wavelength separation property of the photoelectric conversion section. Can be measured. Thereby, the concentration of the sample can be relatively measured using the fluorescence intensity as an index. In these measurements, since the intensity of the scattered light can be taken in as a digital value, the data can be obtained as an image result by the data processor 8 if necessary, and the peak of the scattered light intensity in the sample distribution can be obtained. Various data processing and data display can be performed, such as displaying a value.

Next, an optical scanning mechanism for irradiating the irradiation surface of the sample section 5 with irradiation light will be described. FIG. 2 is a diagram illustrating an optical scanning mechanism that scans with irradiation light using a vibration mirror, and FIG. 3 is a diagram illustrating the relationship between the rotation angle of the vibration mirror and the moving distance of the spot light of irradiation light. In the optical scanning mechanism here, the arrangement positions of the sample unit 5, the light source 21, and the vibrating mirror 22 are in a positional relationship as shown in FIG. When the sample portion 5 is driven to vibrate, the moving speed of the light spot at both ends of the sample portion 5 is changed to the central portion (X =
It becomes faster than near 0). For this reason, the sensitivity detected from the sample part 5 is different between the center part and the end part. On the other hand, in this embodiment, the speed at which the vibrating mirror is driven is corrected and controlled so that the moving speed of the spot light on the irradiation surface of the sample unit 5 becomes constant. That is, the relationship between the position X of the spot light and the angle θ of the mirror is as shown in FIG. 2, and when the distance Z between the center of rotation of the vibrating mirror and the center of the sample unit 5 is used, the mirror angle θ θ is expressed by the following equation. θ = arctan (X / Z) where Z is the distance from the center of rotation of the vibrating mirror 22 to the sample unit 5, and X is the point at which a perpendicular is drawn from the center of rotation of the vibrating mirror 22 to the irradiation surface of the sample unit 5. Is the distance in the surface direction of the gel with respect to the origin.

As a method of correcting the relationship between the rotation angle and the moving distance in this type of optical scanning mechanism, fθ
Although there is a method using a lens, the fθ lens is expensive, and the apparatus for mounting the fθ lens becomes heavy, so here, the mirror driver 30 is provided with a control circuit for variably controlling the rotational angular velocity of the vibrating mirror 22. The correction between the rotation angle of the vibration mirror and the moving distance of the optical scanning mechanism is performed by the correction control of the rotational drive speed of the vibration mirror 22.

FIG. 4 is a block diagram showing a configuration of a main part of a control circuit of a mirror driver for controlling the rotational driving of the oscillating mirror. A galvanometer scanner is used as an actuator of the oscillating mirror, and the rotation angle of the oscillating mirror is controlled by applying a voltage proportional to the rotation angle. In order for the spot light of the irradiation light to move at a constant speed on the irradiation surface of the sample, the distance X of the irradiation surface and the time t may be controlled so as to be in a proportional relationship. Since the relationship between the rotation angle θ of the vibrating mirror and the moving distance X of the spot light is as shown in FIG. 3, the horizontal axis of the graph of FIG. 3 corresponds to the time axis, and the vertical axis corresponds to the voltage axis. A signal having the generated voltage waveform is generated, and is used as a drive control signal for driving the vibrating mirror. Generation of such a drive control signal is performed by a control circuit (30a, 30b, 30c, 30) in the mirror driver 30.
d, 30e), the generated drive control signal is supplied to the actuator of the vibrating mirror 22,
2 is performed.

As shown in FIG. 4, the mirror driver 30 includes a read-only memory 30a storing function waveforms,
A digital / analog conversion circuit 30b for converting the read data of the function waveform into a voltage signal, and a driver 30c for amplifying the converted voltage signal and outputting it as a drive control signal
And a counter 30d for providing a read address to the memory in time series, and an oscillation circuit 30e for providing a clock signal to the counter.

The oscillating circuit 30e operates according to an instruction from the control circuit 27 of the measuring section main body, and a clock signal from the oscillating circuit 30e is input to the counter 30d.
d counts the clock signal, and the read only memory 3
A read address to be supplied to 0a is generated in time series. When the read addresses sequentially generated from the counter 30d are supplied in time series to the read-only memory 30a, the data of the function waveform stored in advance is sequentially read from the read-only memory 30a. In this example, the number of bits of the function data is 12 bits. The read function data is converted into a voltage signal of an analog signal for controlling the rotation angle of the oscillating mirror in the digital / analog conversion circuit 30b. This voltage signal removes the step-like noise by filtering in the driver 30c, further amplifies the power, and as a drive control signal,
It is supplied to the vibrating mirror 22. Thereby, the vibrating mirror can be vibrated at a desired rotation angular velocity such that the moving speed (scan speed) of the spot light of the irradiation light on the sample surface is constant.

The scan speed here is 0.5 Hz, 1 Hz, 2 Hz, 5 Hz, 10 Hz, 20 Hz so as to be approximately logarithmically equal.
It is configured to be able to change at each speed of Hz, 50 Hz, 100 Hz, and 200 Hz. From the control circuit 27 to the mirror driver 30
On the other hand, when the instruction data of the scan speed is sent, the counter 30d and the oscillation circuit 30e are controlled to drive the vibration mirror 22 at a desired scan speed.

FIG. 5 is a diagram showing the configuration of the optical system of the condenser and the photoelectric conversion unit. An example will be described in which the sample section 5 receives a scattered light of the sample in the electrophoresis gel in which the sample is fluorescently labeled and electrophoresed. The main body of the measuring section here is configured so that the gel in which the sample has been injected is sandwiched between glass plates and subjected to electrophoresis, and then the measurement can be performed for the entire glass plate.

The gel 5a of the electrophoresis portion is supported by being sandwiched between glass plates 5b and 5c serving as gel supports. When the irradiation light 31 is irradiated, the irradiation light 31
c, penetrates the gel 5a and the gel support 5b in the thickness direction and reaches the gel 5a. In the gel 5a, the irradiation light 31 travels in the thickness direction. Gel support 5b, 5c
The thickness of the gel 5a is about 5 mm and the thickness of the gel 5a is about 0.35 mm, respectively. Are approximately equal in intensity. Also, the gel 5a and the gel supports 5b, 5c
Irradiation light 31 due to generated light scattering on the light incident surface of
The spread and the decrease in intensity are greatly reduced because the excitation light is incident perpendicularly to the plane in the thickness direction. The irradiation light 31 that has passed through the gel 5a enters the optical trap 32 and is attenuated so as not to have any adverse effect as stray light.

As described above, the scanning of the irradiation light 31 irradiates the gel 5a with the irradiation light 31, and the fluorescence generated from inside the gel 5a by the irradiation of the irradiation light (fluorescence excitation light) is excited. The light is collected by the light collector 23 together with the light scattered by the light itself. The scattered light generated in the gel supports 5b and 5c is geometrically and optically separated by forming an optical path as shown in FIG. 5 by the spatial positional relationship of the light receiving path, and only the fluorescence from the gel is extracted. And sent to the photoelectric conversion unit 24.

In the photoelectric conversion unit 24, scattered light and fluorescence generated in the gel are separated using an optical filter, and weak fluorescence is converted into an electric signal by the photomultiplier. The configuration of the optical path of the condenser 23 is such that the light entrance is one.
The combined optical fiber array 23b is introduced into the photoelectric converter 24.

The configuration of the optical system in the condenser 23 and the photoelectric conversion unit 24 will be described with reference to FIG.
As shown in FIG. 5, an optical path is configured so that the fluorescence from the gel 5a of the sample section 5 and the scattered light of the excitation light generated from the gel supports 5b and 5c are received by the cylindrical lens 23a and collected. I have.

Referring to FIG. 5, the fluorescent light from the sample section 5 and the scattered light of the excitation light generated from the gel supports 5b and 5c reach the cylindrical lens 23a, and the scattered light and the fluorescent light are , Cylindrical lens 2
3a forms an image on the opposite side. Point A in the figure is the focus for the fluorescence from the gel 5a and the scattered light of the excitation light generated from the gel 5a. Also, the gel support 5
In the case of the scattered light of the excitation light generated on the surfaces of the light incident surfaces b and 5c, for example, in the case of the scattered light from the surface of the gel support 5c, an image is formed at point A 'in the figure. Here, the optical fiber array 23b is disposed at the position of the image forming point A so that only the fluorescence from the gel 5a is received at the entrance of the light, so that the optical fiber array 23b is geometrically optically formed by the spatial positional relationship of the light receiving path. Next, the fluorescence is separated from the scattered light from the gel support.

The fluorescent light collected by the entrance (point A) of the optical fiber array is guided into the optical fibers of the fiber array 23 and supplied to the photoelectric conversion unit 24 from the light exits where the optical fibers are bundled. Is done. The fluorescence input to the photoelectric conversion unit 24 is transmitted to the first lens 24a, the aperture 24b, and the second lens 24a.
Only the parallel component is extracted using the lens 24c, and is incident on the optical filter 24d. And the optical filter 2
4d, the scattered light component is removed, and the third lens 24
The light is condensed by e, guided to the photomultiplier tube 24f, and the detected fluorescence is converted into an electric signal.

In the photoelectric conversion unit 24, an optical filter 24d
The incident light for improving the wavelength separation property of the optical filter 24d is converted into only a parallel light component by the second lens, and the optical filter 24d
At a right angle. Then, the scattered light of the excitation light generated in the gel is separated by the optical filter 24d,
The signal-to-noise ratio is improved, and the light is condensed by the third lens 24e and guided to the photomultiplier tube 24f.

After the fluorescent light is received and condensed as described above and the scattered light is removed by the optical filter, the fluorescent light guided to the photomultiplier tube 24f is converted into an electric signal by the photomultiplier tube 24f. Is output. Photomultiplier tube 2
The electric signal output from 4f is input to the amplifier 25. Then, in the amplifier 25, the weak signal is sufficiently amplified by the amplification stage including the integration circuit.

When a measurement is performed using a thin film filter such as a nylon membrane or a nitrocellulose membrane as the support, the gel 5a is used instead of the gel 5a in the above-described measurement of the electrophoresis gel, and the support on which the sample is adsorbed is used. What is necessary is just to sandwich a body thin film filter. In this case, the light from the thin-film filter is almost reflected light with respect to the irradiation of the irradiation light 31. However, by setting the light receiving surface (A) as described above, the optical axis of the light receiving surface is different from the main axis of the reflected light. Since the light receiving surface is different, the light receiving surface does not directly receive the reflected light, and a sufficient detection sensitivity can be obtained by suppressing noise.

In this case, as in the case of reading the fluorescence pattern of the sample labeled on the electrophoresis gel, glass plates are used as the supports 5b and 5c of the sample portion 5, but the supports 5b and 5c The scattered light reflected by the sample is removed by the cylindrical lens 23a, and by changing the optical filter 24d in accordance with the wavelength of the scattered light of the sample on the thin film filter, the relative light such as the concentration or absorbance of the sample is directly changed. You can make measurements. Furthermore,
The absorbance can be measured even on the support of such a thin film filter, and the concentration, the absorbance, and the like can be measured even on a sample obtained by fluorescently labeling a sample.

FIG. 6 is a circuit diagram showing a configuration of an amplifier including an integrating circuit. As shown in FIG. 6, the amplifier 25 of the main body of the measuring section is provided with an integrating circuit composed of an operational amplifier 25a at the preceding stage, and an output amplifier circuit composed of the operational amplifier 25b at the next stage. It constitutes an integral amplification stage. The electric signal from the photomultiplier tube 24f is input to the operational amplifier 25a. The operational amplifier 25a constitutes an integrating circuit together with the capacitor 25c and the switch 25d for controlling the integrating operation. The output of the integrating circuit is input to the subsequent operational amplifier 25b and amplifies the output at a gain determined by an external resistor. Is sent to the next analog-to-digital conversion circuit.

Next, the operation of the amplifier 25 including the integrating circuit thus configured will be described with reference to the timing chart of FIG. Since the output of the photomultiplier tube 24f of the photoelectric conversion unit 24 has a very large output impedance, it can be almost regarded as a current source. The operational amplifier 25a has a high input impedance of FET (field effect transistor) input type.
When the switch 25d is turned off, the entire output current ip of the photomultiplier tube 24f becomes the current flowing through the capacitor 25c as it is. With this current, the output voltage of the operational amplifier 25a becomes a ramp function output as shown in FIG. In the integration operation of the amplifier 25, integration is performed for a time corresponding to one pixel, and a sampling circuit in the analog-to-digital conversion circuit 26 performs sampling in accordance with the timing of the S / H clock, holds it as it is, -The digital conversion circuit 26 converts the signal into a digital signal.

After being held, the charge stored in the capacitor 25c is discharged by activating a C / D clock which is a C / D control signal applied to the switch 25d. Hereinafter, similarly, the integration operation for such a time corresponding to one pixel is repeated. An amplification stage using an integration circuit using such an operational amplifier differs from a pseudo integration circuit consisting only of a resistor and a capacitor, in that the charge from the photomultiplier tube 24f can be almost completely integrated. Therefore, a high signal-to-noise ratio can be obtained. Also, the integration time can be arbitrarily changed by changing the C / D clock of the C / D control signal for the switch 25d. Therefore, it is possible to easily adjust the degree of amplification for amplifying the weak signal comprehensively.

In the case of this example, by synchronizing with the scanning operation of the mirror driver 30 shown in FIG. 4, it is possible to control according to the size of the area of the sample to be read, and to reduce the dead time of reading. Can be eliminated. Irradiation light (excitation light) is adjusted according to the intensity of fluorescence from the sample.
Since the scan speed and the integration time of the light-receiving-side amplifier can be freely set, a very flexible device can be configured.

[0051]

As described above, according to the densitometer of the present invention, two-dimensional measurement can be performed by the optical scanning mechanism without depending on the state of the sample spot, and the spatial position of the light receiving path can be measured. Sample and sample adhered
By selectively receiving the scattered light from the sample support , the absorbance of the sample can be measured. In addition, by using the densitometer of the present invention, it is possible to measure the distribution of a fluorescent substance in which a sample is fluorescently labeled, in addition to the measurement of the absorbance.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a configuration of a main part of a measuring apparatus main body of a densitometer according to one embodiment of the present invention;

FIG. 2 is a diagram illustrating an optical scanning mechanism that scans with irradiation light using a vibrating mirror;

FIG. 3 is a diagram for explaining a relationship between a rotation angle of a vibrating mirror and a moving distance of a spot light of a laser beam;

FIG. 4 is a block diagram illustrating a configuration of a main part of a control circuit of a mirror driver that controls rotational driving of a vibrating mirror;

FIG. 5 is a diagram showing a detailed configuration of an optical system of a condenser and a photoelectric conversion unit;

FIG. 6 is a circuit diagram showing a circuit configuration of an amplifier including an integration circuit;

FIG. 7 is a time chart showing the timing of a read operation of the amplifier.

[Explanation of symbols]

 5 Sample Unit 8 Data Processor 13 Scattered Light 21 Light Source 22 Vibrating Mirror 23 Concentrator 24 Photoelectric Converter 25 Amplifier 26 Analog-to-Digital Conversion Circuit 27 Control Circuit 28 Storage Circuit 29 ... Interface 30 ... Mirror driver 31 ... Laser beam 32 ... Optical trap 44 ... Folding mirror

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Naganori Nasu 6-81 Onoecho, Naka-ku, Yokohama-shi, Kanagawa Prefecture Within Hitachi Software Engineering Co., Ltd. (56) References JP-A-51-23795 (JP, A) JP-A-2-242144 (JP, A) JP-A-1-140067 (JP, A) JP-A-3-295463 (JP, A) JP-T-61-502352 (JP, A) (58) Fields investigated (Int.Cl. ⁶ , DB name) G01N 21/00-21/01 G01N 21/17-21/61 G01N 27/447

Claims (4)

(57) [Claims] A sample is adsorbed or adhered to a sample support having a light scattering property, and the sample is dyed or labeled as it is or by using a specific substance on the sample.
By scanning the sample support is irradiated, the measuring a reflected light from the sample support and sample, a densitometer for measuring the absorbance of the sample or substance, a light source for emitting the illumination light, the illumination light An optical scanning mechanism that scans the irradiation light in the thickness direction of the sample support by shaking the optical axis, and sets a light receiving surface in a direction different from the optical axis of the irradiation light, and applies the irradiation light to the sample support. Arrange on the side to irradiate,
A light receiving unit that selectively receives scattered light from the sample support and the sample based on a spatial positional relationship with the light receiving path; a photoelectric conversion unit that photoelectrically converts an optical signal received by the light receiving unit and outputs an electric signal; It is characterized by comprising a data acquisition unit that acquires data corresponding to the scanning of irradiation light by the optical scanning mechanism with respect to the electric signal from the photoelectric conversion unit, and a data processing unit that calculates the absorbance of the sample based on the acquired data. Densitometer. 2. A sample support is adsorbed or adhered to a sample support having light scattering properties, and the sample is stained or labeled as it is or by using a specific substance on the sample, and irradiation light is scanned on the sample support. A densitometer for measuring the reflected light from the sample support and measuring the absorbance of the sample or the substance, wherein the light source emits irradiation light, and the optical axis of the irradiation light is shaken to support the sample. A light scanning mechanism that scans the irradiation light in the thickness direction of the body , a light receiving surface is set in a direction different from the optical axis of the irradiation light, and the light receiving surface is arranged on the side that irradiates the sample support with the irradiation light,
The optical axis of the light receiving section intersects the optical axis of the irradiation light on the sample surface, and selectively receives scattered light from the sample support and the sample based on a spatial positional relationship with the light receiving path. A photoelectric conversion unit that photoelectrically converts a received optical signal and outputs an electric signal; and a data acquisition unit that amplifies the electric signal from the photoelectric conversion unit in correspondence with scanning of irradiation light and acquires data from the electric signal. And a data processing unit for calculating the absorbance of the sample based on the acquired data. 3. The densitometer according to claim 1, wherein the light source is a laser, and the substance for staining or labeling the sample is Coomassie brilliant blue or an analog thereof, amide black or silver stain, or gold. A densitometer using at least one of colloidal staining. 4. The densitometer according to claim 1, wherein a fluorescent substance labeled on a sample adsorbed or adhered to a sample support having light scattering properties is excited by irradiation light to emit fluorescence. A densitometer for measuring scattered light from a sample and a sample support, measuring the distribution of absorbance thereof, and reading a fluorescent pattern emitted from the sample.