JPH0918772A - Microscopic image analyzer - Google Patents

Abstract

PURPOSE: To provide the microscopic image analyzer by which an optional point or an optional area in a 2-dimensional image is measured at a high speed and a timewise change in a light quantity from a sample is measured at a high speed. CONSTITUTION: The microscopic image analyzer fitted to a microscope 1 to measure a change over time in a 2-dimensional luminous quantity distribution from a sample 7 is provided with a 2-dimensional solid-state image pickup element 10 accessing data from an optional image in an optional order, a picture element address designation means 11 designating an optional picture element from a 2-dimensional image of the element 10, an address storage means 12 storing a read address designated by the means 11, a clock generating means 13 outputting sequentially an address stored in the means 12, and an A/D conversion means 14 converting an output from the microscope 1 into a digital signal.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope image analysis apparatus which can be attached to a microscope and can measure a change with time of a two-dimensional light amount distribution from a sample.

[0002]

2. Description of the Related Art Conventionally, it has been practiced to measure the state, components, properties of a sample or their changes over time from changes in the amount of light in a microscope image. In particular, in the field of biology, a sample is stained with a fluorescent dye, and by irradiating this with excitation light, an image of the generated fluorescence is captured, and the intensity of the image or a plurality of excitation wavelengths or fluorescence wavelengths is used. A fluorescence microscope apparatus for measuring properties in a sample from an image obtained by performing a calculation between images is known (Japanese Patent Laid-Open No. 2-2854).
No. 2).

Generally, when measuring a physiological phenomenon of a cell using a fluorescence microscope, since the amount of light in an image is small and the change over time is large, (1) high sensitivity and signal noise ratio ( The S / N ratio must be high, and (2) the condition that an image can be captured at high speed must be satisfied. However, when a video signal obtained by a general image pickup device is stored or processed to obtain data, the measurement time resolution is 1/60 second to 1 /
It will depend on the speed of the video signal of 30 seconds.

Conventionally, there has been no fluorescence microscope apparatus satisfying all the above conditions, but an example thereof will be described below. <
First example> As a condition that satisfies the condition of (2), for example, as disclosed in Japanese Patent Laid-Open No. 293650/1990, a television camera capable of raster scanning and random scanning is used to detect a specific position. There are devices that perform high-speed measurement by measuring only data. In this case, an image dissector that reads data at an arbitrary position at high speed is used as an image sensor.

<Second Example> As an example of improving the sensitivity in the condition (1), there is a device using a CCD (solid-state image sensor) camera with an image intensifier, for example, the CCD and the image intensifier. (When an image is formed on the photocathode, an amount of photoelectrons is generated according to the brightness of the image).

<Third Example> S / N of the conditions of (1)
A device having a high ratio is a device using a cooled CCD camera, which is a device in which a CCD device is electronically cooled to reduce a dark current.

<Fourth Example> In order to improve the problem of the third example, that is, low speed, data of only a certain block is read.

[0008]

The conventional example described above has the following problems. In the first example, the image dissector sweeps a straight line connecting two points to obtain data at two points, and obtains data at each point. , Need unnecessary sweep time. Therefore, when the measurement points are scattered on the two-dimensional image or when the measurement points are distant from each other, there is a drawback that it takes time to acquire the data.
In addition, the image dissector does not have a storage function because of its structure, unlike an ordinary image sensor, and thus the signal noise ratio (S
There is also a problem that the / N ratio is bad. Furthermore, in a normal image sensor, such as an image pickup tube or a solid-state image sensor, the data of any pixel is randomly ordered because of its structure.
Is difficult to read with.

In the second example, the CCD with an image intensifier has high sensitivity, but the S / N ratio is normal, and the speed is usually 30 frames per second (33 msec / frame), which is on the order of msec. It is impossible to respond to changes in

In the third example, the S / N is high, but the speed is further inferior to the CCD with an image intensifier. In the fourth example, it is difficult to randomly read the data of any element from the entire element (at a high speed only for necessary data).

An object of the present invention is to provide a microscope image analysis apparatus capable of measuring any point or area in a two-dimensional image at high speed and measuring the time change of the light quantity from the sample at high speed. .

[0012]

In order to achieve the above object, the invention according to claim 1 is mounted on a microscope,
A two-dimensional solid-state image sensor capable of accessing data from an arbitrary pixel in an arbitrary order in a microscope image analyzer for measuring a temporal change of a two-dimensional light amount distribution from a sample, and
Pixel address designating means for designating an arbitrary pixel of the two-dimensional image from the three-dimensional solid-state image pickup device, address storage means for storing the read address designated by the pixel address designating means, and address stored in the address storage means. Address generating means for sequentially outputting the two-dimensional solid-state image sensor to the two-dimensional solid-state image sensor, and A / D conversion means for digitally converting the output from the two-dimensional solid-state image sensor in synchronization with the output timing of the sequentially output addresses. And a data processing means for processing the luminance data obtained by the A / D conversion means, and a microscope image analysis apparatus.

In order to achieve the above-mentioned object, the invention according to claim 2 is a microscope image analyzing apparatus which is attached to a microscope to measure a change with time of a two-dimensional light amount distribution from a sample. A two-dimensional solid-state image sensor that can access data in order, a pixel address designating unit that designates an arbitrary pixel of a two-dimensional image from the two-dimensional solid-state image sensing unit, and a read address designated by the pixel address designating unit. Address storing means for storing, address generating means for sequentially outputting the addresses stored in the address storing means to the two-dimensional solid-state imaging device,
A / D conversion means for digitally converting the output from the two-dimensional solid-state imaging device in synchronization with the output timing of the sequentially output addresses, and the wavelength of the light source can be switched to a plurality of wavelengths. A microscope image analysis apparatus comprising: an illumination wavelength switching means for driving the address generating means in synchronization.

In order to achieve the above object, the invention according to claim 3 provides the microscope image analyzing apparatus according to claim 1 or 2 with an amplifying means for amplifying an input signal of the two-dimensional solid-state image pickup device. It is a microscope image analysis device.

[0015]

According to the invention according to claim 1, since the two-dimensional solid-state image pickup device capable of accessing the data from any image in any order is used, any point or area in the two-dimensional image can be detected at high speed. Therefore, it is possible to measure the change with time of the amount of light from the sample at high speed.

According to the invention corresponding to claim 2, in addition to the operation of claim 1, an illumination wavelength switching means is provided.
When measuring the intracellular ion concentration, the illumination wavelength switching means sequentially switches a plurality of illumination light wavelengths to measure the amount of fluorescence at each wavelength, and the ion amount can be obtained by dividing these. .

According to the invention corresponding to claim 3, in addition to the operation of claim 1 or claim 2, the image intensifier is provided in front of the two-dimensional solid-state image pickup device.
Even dark samples such as faint reflected light images and fluorescent images can be measured.

[0018]

Embodiments of the present invention will be described below with reference to the drawings. <First Embodiment> FIG. 1 is a block diagram showing a first embodiment of the present invention, which is a microscope 1, a two-dimensional solid-state image pickup device 10, a pixel address designating means 11, an address storing means 12, a clock generating means. The clock generator 13 includes an A / D converter 14, a data processor 15, a display 16, and a memory address controller 17.
The memory address control means 17 constitutes the address generation means of the present invention.

The microscope 1 comprises a light source 2, lenses 3 and 4, a half mirror 5 and an objective lens 6, and an image of transmitted light, reflected light or fluorescence from a sample 7 illuminated by the light source 2 is transmitted through the objective lens 6. An image is formed on the two-dimensional solid-state image sensor 10.

The two-dimensional solid-state image pickup device 10 can access (randomly access) data from an arbitrary image in an arbitrary order, for example, a well-known internal amplification type solid-state image pickup device (AMI: Amplified Mos Image).
r).

As shown in FIG. 2, this AMI basically amplifies an optical information signal obtained by a photoelectric conversion unit composed of a plurality of pixels (single cell for light reception and amplification) 101 in the same pixel, Vertical and horizontal scan switch circuits 102, 10
It is an XY address type that is read via

As shown in FIG. 3, one of the pixels 101 is a photodiode D as a photoelectric conversion unit, and three n-channel MOSFEs for a reset transistor Trs, an amplification transistor Ta and a vertical selection transistor Ty.
It is composed of T. The horizontal switch Tx is provided for each vertical signal line.

In principle, the photodiode D is reverse-biased, discharged by photocharge, and its potential is amplified by current and taken out. This operation is as follows. In the reset period, the photodiode D is set to the initial value (positive potential) Vrs via the reset transistor Trs. In the accumulation period, of the electrons and hole pairs excited by the irradiation of light, the electrons are the capacitance C of the photodiode D.
The holes are accumulated in the PD and the holes flow out to the substrate. Therefore, the potential of the photodiode D decreases according to the incident light. This potential is applied to the gate of the amplification transistor Ta, and the amplified current corresponding to the potential of the photodiode D is read out via the vertical scanning switch Ty and the horizontal scanning switch Tx. In addition, Xi, Yn, and Yn + 1 are terminals, Vs is an output voltage, I
S is the output current, RL is the load resistance, and Vp is the gate potential.

In the AMI described above, since the photoelectric conversion section and the amplification section are extremely close to each other, there is no mixing of noise and no signal attenuation, and ideal amplification can be performed, so that an output with good S / N can be obtained. The pixel addressing means 11 is a two-dimensional solid-state image sensor 1.
Designate an arbitrary pixel from the two-dimensional image from 0. The address storage unit 12 is composed of a memory circuit, and the read address designated by the pixel address designating unit 11 is sequentially stored in each memory address. For example, when it is desired to acquire a two-dimensional image of the entire image, it is possible to set the address storage unit 12 from the top pixel address to the last address in order.

The clock generation means 13 sequentially outputs the addresses stored in the address storage means 12. The A / D conversion unit 14 converts the output from the two-dimensional solid-state imaging device 10 into a digital signal in synchronization with the clock generation unit 13. The data processing unit 15 converts the converted digital data into image processing or various data processing, for example, brightness or color matching the numerical value of the data, and displays it on the display unit 16. The memory address control means 17 repeatedly generates from the head memory address to a valid memory address in synchronization with the clock signal generated from the clock generation means 13.

According to the first embodiment constructed as described above, the following operational effects can be obtained. That is, in each pixel 101 of the two-dimensional solid-state imaging device 10, the input light is converted into an electric quantity according to the light quantity. Each pixel 1
A pixel address is assigned to 01, and when this pixel address is specified in the address storage means 12, the specified pixel 101 outputs an electric quantity corresponding to the light quantity, for example, a voltage or a current.

Then, the memory address control means 17 repeatedly generates from the head memory address to a valid memory address in synchronization with the clock signal generated from the clock generation means 13.

As described above, the two-dimensional solid-state image pickup device 10 outputs an electric signal corresponding to the light amount of each pixel set in synchronization with the clock signal. The output electric signal is converted by the A / D conversion means 14 into a digital signal synchronized with the clock generation means 13. The converted digital signal is subjected to various data processing by the data processing means 15 and drawn and visualized on the display means 16. According to such an apparatus, it is possible to realize high-speed collection of data of an arbitrary point or area in the following two-dimensional image.

As the sample, for example, consider a case where cells scattered in the image are stained with a fluorescent dye as shown in FIG. Here, consider a fluorescent image when a short arc lamp such as a high pressure mercury lamp or a xenon lamp is used as the light source 2 in FIG. 1 and a sample is illuminated.

First, the pixel address designating means 11 stores the addresses of all the pixels in the address storing means 12.
As a result, the two-dimensional solid-state image sensor 1 is displayed on the display unit 16.
The two-dimensional image in which 0 is captured is displayed repeatedly. In this state, the focus of the sample 6 can be adjusted and the measurement position can be adjusted. The pixel to be measured is specified from the displayed image by the following procedure.

One or more regions containing the target cells are designated in the two-dimensional image. The specification method is the diagonal of a rectangle. 2
Specify a point. Specify an ellipse inscribed in this rectangle,
It is possible to specify the center and radius of the circle, specify the inside surrounded by multiple straight lines, and specify the inside surrounded by a free-form curve. Next, a threshold value is set inside the area designated by these, and the address of the pixel that is equal to or greater than this threshold value is stored in the pixel address storage means 12 as the measurement target. After that, the data can be read at only the pixel address to be measured, so that the measurement can be performed at high speed.

<Second Embodiment> As shown in FIG. 5, the difference from the first embodiment of FIG. 1 is that between the light source 2 and the lens 3, the illumination wavelength switching means 20 for switching the wavelength of the light source 2 at high speed. Is provided and a switching signal obtained from the illumination wavelength switching means 20 is introduced into the clock generating means 13.

As an example of the illumination wavelength switching means 20, there is one configured as shown in Japanese Patent Laid-Open No. 6-160722, for example, and FIG. 6 is for explaining this. A lamp house 22 having a light source 31 that emits excitation light is attached by the attachment mechanism 21. The lamp house 2 is located at the focal position of the collector lens 23 (focal length f).
A mirror 24 for deflecting the light emitted from the optical disc 2 at a desired angle with respect to the incident optical axis is arranged by a mirror driving device 25 so that the surface direction can be changed. The reflected light from the mirror 24 enters the collimator lens 27 through the opening of the light shielding plate 26 according to the direction of the mirror 24, and the interference filter 28.
And then enters the collector lens 29. At the focal point of the collector lens 35 (focal length f), the mounting mechanism 34
Thus, the end surface of the light guide 36 attached to the apparatus body is located. The interference filter 28 is attached to the slider 29 and can be easily attached to and detached from the guide rail 30 of the apparatus body. Further, behind the collector lens 23, there is provided a shutter 32 for blocking the light incident from the lamp house 22 as necessary. A light blocking tube 33 is provided between the collimator lens 27 and the interference filter 28 and between the interference filter 28 and the collector lens 35.

The illumination wavelength switching means 20 described above constitutes a deflecting means with the mirror 24 and the mirror driving device 25. By changing the direction of the mirror 24, desired light is made incident and the light guide 36 is guided. The wavelength of the emitted illumination light can be changed.

According to the second embodiment constructed as above, the following operational effects can be obtained. That is, when measuring the intracellular ion concentration, the illumination wavelength switching means 2
With 0, a plurality of illumination light wavelengths are sequentially switched, the fluorescence amount at each wavelength is measured, and the ion amount can be obtained by dividing these.

In this case, it takes a finite time to switch the wavelength of the illumination light, so that there is an effective output signal in the output from the image pickup device 10 and an invalid output signal during the switching. .

From the above, in the present embodiment, the wavelength switching from the illumination wavelength switching means 20 is completed, and a signal indicating that the illumination light is in an effective state is introduced into the clock generating means 13, and when the signal is effective. Since only the clock signal is output, only the effective output from the image sensor 10 can be taken out.

As a modification of the second embodiment, the wavelength of the illumination wavelength switching means 20 is not switched while the effective pixel addresses from the beginning to the end are output from the address storage means 12. The switching prohibition signal may be output to the illumination wavelength switching means 20.

<Third Embodiment> As shown in FIG. 7, in front of the two-dimensional solid-state image pickup device 10, an image intensifier 40 composed of an electron lens for accelerating and imaging the photoelectrons generated from the photoelectric conversion surface is provided. Only the point provided is the second of FIG.
This is a difference from the embodiment.

As shown in FIG. 8, the image intensifier 40 has a photoelectric conversion surface 41 and an electron lens 42 for accelerating and forming an image of photoelectrons generated from the photoelectric conversion surface 41.
And a microchannel plate 43 and a fluorescent screen 44. The microchannel plate 43 has innumerable thin holes (channels), and when electrons enter the holes, the electrons are attracted by a potential gradient and the electrons collide with the inner wall several tens of times and come out from the opposite side. Since secondary electrons are emitted from the wall surface due to collision of electrons, output electrons are magnified several thousand times or more with respect to incident electrons. As a result, each channel of the micro channel plate 43 corresponds to a pixel, Each pixel is enhanced at the same time.

According to the third embodiment thus constructed,
The following operation and effect can be obtained. The above-mentioned first and second
In the embodiment, the two-dimensional solid-state image pickup device 1 is used to directly obtain a microscope image.
An example in which the output from the image sensor 10 is measured at this time by imaging on 0, but in this case, when measuring weak reflected light from the sample 7 or a fluorescent image, the sensitivity is insufficient. It is possible. However, in the third embodiment, the image sensor 1
Since the image intensifier 40 is provided in front of 0, not only can the arbitrary point or region in the two-dimensional image be measured at high speed as in the above-described embodiment, but also in addition to the effect of the above-described embodiment. , Faint reflected light image, fluorescent image,
Even dark sample 7 can be measured.

<Modification> In the above embodiment, the AMI is taken as an example of the two-dimensional solid-state image pickup device 10, but
In addition to MI, for example, CMD, photodiode array (arrangement of photodiodes in a matrix), frame transfer CCD (solid-state image sensor CCD data is collectively transferred to a memory circuit, and the contents are sequentially read. The same effects as those of the above-described embodiment can be obtained.

Further, as the illumination wavelength switching means, the one configured as shown in FIG. 6 has been mentioned, but other than this, a means for switching by rotating a plurality of disc-shaped filters, an optical chopper / sector mirror, etc. are used. The same effect can be obtained by using a galvanometer scanner.

[0044]

According to the present invention, it is possible to provide a microscope image analysis apparatus capable of measuring any point or area in a two-dimensional image at high speed and measuring the time change of the light quantity from the sample at high speed. You can

[Brief description of the drawings]

FIG. 1 is a block diagram for explaining a first embodiment of a microscope image analysis apparatus of the present invention.

FIG. 2 is a diagram for explaining an example of the two-dimensional solid-state image sensor 10 of FIG.

FIG. 3 is an equivalent circuit diagram for explaining one pixel in FIG.

FIG. 4 is a view showing an example of the sample of FIG.

FIG. 5 is a block diagram for explaining a second embodiment of the microscope image analysis apparatus of the present invention.

FIG. 6 is a diagram for explaining an example of the illumination wavelength switching means of FIG.

FIG. 7 is a block diagram for explaining a third embodiment of the microscope image analysis apparatus of the present invention.

FIG. 8 is a diagram for explaining an example of the image intensifier shown in FIG. 7.

[Explanation of Codes] 1 ... Microscope, 2 ... Light source, 3, 4 ... Lens, 5 ... Half mirror, 6 ... Objective lens, 7 ... Sample, 10 ... Two-dimensional solid-state imaging device, 101 ... Pixel, 11 ... Pixel addressing means,
12 ... Address storage means, 13 ... Clock generation means, 1
4 ... A / D conversion means, 15 ... Data processing means, 16 ... Display means, 17 ... Memory address control means, 20 ... Illumination wavelength switching means, 40 ... Image intensifier.

Claims (3)

[Claims] 1. A two-dimensional solid-state image sensor capable of accessing data from any pixel in any order in a microscope image analysis apparatus which is attached to a microscope to measure a change with time of a two-dimensional light amount distribution from a sample. Pixel address designating means for designating an arbitrary pixel of the two-dimensional image from the two-dimensional solid-state image pickup device, address storing means for storing the read address designated by the pixel address designating means, and this address storing means. Address generating means for sequentially outputting different addresses to the two-dimensional solid-state imaging device, and A / D conversion for digitally converting the output from the two-dimensional solid-state imaging device in synchronization with the output timing of the sequentially output addresses. Means and data processing means for processing the brightness data obtained by the A / D conversion means, Analysis apparatus. 2. A two-dimensional solid-state image sensor capable of accessing data from any pixel in any order in a microscope image analyzing apparatus which is attached to a microscope to measure a change with time of a two-dimensional light amount distribution from a sample. Pixel address designating means for designating an arbitrary pixel of the two-dimensional image from the two-dimensional solid-state image pickup device, address storing means for storing the read address designated by the pixel address designating means, and this address storing means. Address generating means for sequentially outputting different addresses to the two-dimensional solid-state imaging device, and A / D conversion for digitally converting the output from the two-dimensional solid-state imaging device in synchronization with the output timing of the sequentially output addresses. Means, and the wavelength of the light source can be switched to a plurality of wavelengths, and the address generating means is driven in synchronization with this switching. A microscope image analysis device comprising: an illumination wavelength switching unit that allows 3. The microscope image analyzing apparatus according to claim 1, further comprising an amplifying unit that amplifies an input signal of the two-dimensional solid-state image pickup device.