JPH09127419A - Image processing circuit for scan type microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce a circuit scale and to make possible quantizing an optical signal of plural channels much by photoelectrically converting sample light, amplifying at every plural channels, circularly selecting in time division and controlling the distributing timing. SOLUTION: Photoelectric conversion circuits 31a-31c photoelectric convert sample light at every channel to amplify, and sample-and-hold circuits 33a, 33b hold respectively signals of other channels excepting one channel among the amplified signals. Then, a data selector 34 selects circularly the signals of the held channels and the signal of the not held channel in time division to quantize by an A/D converter 35. The data selector 36 distributes and selects the quantized signals according to the channels to store the distributed signals in memories 37a-37c at every channel. At this time, a sample clock generation circuit 38 supplies an operation clock to the sample-and-hold circuits 33a, 33b, the data selectors 34, 36 and the A/D converter 35 to control the timing.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an image processing circuit applied to a scanning microscope for detecting a light from a sample obtained by two-dimensionally scanning a sample with a light beam to obtain a sample image.

[0002]

2. Description of the Related Art An optical microscope has a structure in which a sample on a slide placed on a stage is magnified and observed with an objective lens. Generally, for illumination of a sample, light from a light source such as a lamp is used with a condenser lens. Therefore, the entire observation area of the specimen is uniformly illuminated.

However, when such a structure is adopted as the illumination system, there are problems such as flare and it is very difficult to observe a low-contrast sample. In order to improve these problems, a scanning optical microscope which is a point light projection type (spot light projection type) optical microscope has been proposed.

This optical microscope irradiates a point light source such as a laser light source on an observation sample through an objective lens in a point-like manner, thereby irradiating transmitted light or reflected light of the observation specimen or point-like light to the specimen. The fluorescent light generated from the above is again imaged in a point shape via the objective lens and the optical system, and this is detected by the detector to obtain the grayscale information of the image.

However, with this alone, only the grayscale information of only the point position illuminated by the point light source can be obtained.
The sample and the irradiation point position are relatively two-dimensionally scanned, such as an XY scanning method of moving in the two-dimensional plane by moving in the directions of the axis and the Y-axis, and an optical system for scanning the optical path. ,
By displaying a bright point corresponding to the signal of the density information at each point position corresponding to the XY scanning position by a display device such as a CRT display in synchronization with the XY scanning, an image can be observed. I have to.

The above is the principle configuration of the scanning microscope. Specifically, in general, the scanning microscope generally detects transmitted light, reflected light, or fluorescence from a sample scanned with a laser beam as a photoelectron multiplier. A photoelectric signal processing circuit that further quantizes by an A / D conversion circuit and stores it in a memory is provided in order to hold image data converted into an electric signal by a photoelectric converter such as a double tube or a photodiode.

FIG. 4 shows an example of a typical conventional configuration in the process of quantizing the photoelectrically converted analog signal in the scanning microscope. This drawing refers to the Institute of Electronics and Communication Engineers Technical Research Report Meeting SSD 79-56 "Laser Scanning Device Analysis System", and here, explanation will be made by omitting 3 channels into 2 channels. This is, for example, 2
A plurality of light receiving sections are provided so that two types of fluorescence having different wavelengths can be simultaneously detected by using different types of fluorescent dyes.

In the figure, the separated two channels (CH1,
The light of CH2) is photoelectrically converted by the photoelectric conversion circuits 1a and 1b, and the obtained analog electric signals are amplified by the amplifier circuits 2a and 2b, respectively. Each of these amplified signals is supplied to A / A at a timing synchronized with the clock of the sample clock circuit 4.
It is quantized by the D converters 3a and 3b, converted into digital values, and then stored in the memories 5a and 5b.

Therefore, the circuit configuration as shown in FIG. 4 has the same number of A / D converters as the number of channels,
It quantizes multiple channels at the same timing.

[0010]

However, FIG.
In the circuit configuration as shown in FIG. 3, since individual A / D converters are used for each channel as described above, A / D converters are required for the number of channels, and the overall circuit scale is large and expensive. It will be something like.

Strictly speaking, each A / D converter has its own characteristic error in A / D conversion. When a plurality of A / D converters are used in parallel, the characteristic error and the like are also taken into consideration. Must.

The present invention has been made in view of the above circumstances, and an object of the present invention is to reduce the number of A / D converters used as much as possible to reduce the circuit scale and to reduce the A / D converter.
An object of the present invention is to provide an image processing circuit for a scanning microscope capable of quantizing optical signals for a plurality of channels and storing the signals for each channel without being affected by the characteristic error of the converter.

[0013]

According to the first aspect of the present invention,
In a scanning optical microscope, which detects light from the sample obtained by two-dimensionally scanning the sample with a light beam and processes the light to obtain a sample image, a plurality of channels for photoelectrically converting the light from the sample A photoelectric conversion unit provided for each of the units, an amplification unit that amplifies each signal of an analog value obtained by the photoelectric conversion unit for each of a plurality of channels, and one of the signals amplified by the amplification unit except one channel Holding means for respectively holding the signals of the respective channels, and first signal selecting means for cyclically selecting, in a time division manner, the signals of the channels held by the holding means and the signals of the channels not held by the holding means. , A converting means for converting an analog value signal sent via the first signal selecting means into a digital value, and a signal converted by the converting means into a digital value, Second selecting sorting according to Yan'neru
Signal selecting means, storage means for storing the signal of each channel distributed and selected by the second signal selecting means for each channel, the holding means, the first signal selecting means, the converting means and the second means. The signal selecting means is provided with a control means for supplying an operation clock to perform timing control.

As a result, one A / D converter constituting the above conversion means is time-division driven to quantize optical signals for a plurality of channels, which can be stored for each channel. Only one A / D converter is required to quantize a signal, and the overall circuit scale can be significantly reduced, and at the same time, the error in the characteristics at the time of quantization with respect to optical signals for a plurality of channels can be reduced. Is similar,
It is possible to unify the signal characteristics among the channels.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION An embodiment in which the present invention is applied to a scanning optical microscope apparatus will be described below with reference to the drawings. FIG. 1 shows its mechanical and optical structure. The laser beam emitted from the laser light source 11 is appropriately focused by the spot projection lens 12, reflected by the dichroic mirror 13, and further rotated by a galvanometer rotating about a rotation axis in a direction orthogonal to the plane of the drawing. After being reflected by the mirror 14, an image is formed on the objective image plane by the pupil projection lens 15, and thereafter, it is incident on the optical path switching mirror 16.

Therefore, the laser beam is scanned by the rotating galvanometer mirror 14 at the position of the optical path switching mirror 16 in the vertical direction in the figure. The laser beam reflected by the optical path switching mirror 16 enters the objective lens 17 and is imaged on the sample 18. Therefore, the laser beam imaged on the sample 18 is scanned on the surface of the sample 18 in the left-right direction in the drawing.

The laser beam is scanned in the left-right direction by the optical deflection irradiation by the galvanometer mirror 14, and at the same time, the specimen 18 is scanned.
By moving and scanning the scanning stage 19 on which is mounted in the direction orthogonal to the paper surface of the drawing, the surface of the sample 18 can be two-dimensionally scanned with a laser beam.

The fluorescent dye pre-biochemically labeled on the cells on the specimen 18 is excited by this laser beam to emit fluorescence. The fluorescence emitted from the sample 18 traces back the above-mentioned optical path in reverse, and the objective lens 17, the optical path switching mirror 16, the pupil projection lens 15, and the galvanometer mirror 14 are taken.
After that, the light is transmitted through the dichroic mirror 13 upward in the drawing.

The fluorescence transmitted through the dichroic mirror 13 is separated by the second dichroic mirror 20 into its wavelength components. The separated fluorescent light passes through the barrier filters 21a and 21b, respectively, and the condenser lens 22
The light is condensed on the light receiving surfaces of the photomultiplier tubes 23a and 23b by a and 22b.

On the other hand, the scattered light scattered by the cells on the specimen 18 is condensed by the condenser lens 24 together with the light transmitted downward through the specimen 18, and the beam splitter 25
It is incident on the ring slit 26 after being reflected by.

The ring slit 26 blocks the transmitted light from the sample 18, transmits only the scattered light, and makes it incident on the light receiving surface of the photodiode 27. The optical path switching mirror 16 is provided so that it can be inserted into and removed from the microscope body. By removing the optical path switching mirror 16, the image of the sample 18 can be guided to the observation optical system 28.
By providing the observer with illumination by the transmitted illumination optical system 29 and the coaxial incident illumination optical system 30, the transmitted image and the reflected image of the specimen 18 can be observed with the naked eye with a microscope, or can be photographed with a television camera or a photographing device. be able to.

In the scanning optical microscope apparatus having such a mechanical and optical structure, the specimen 18 is two-dimensionally scanned with a laser beam, and the fluorescence and scattered light obtained from the specimen 18 are converted into photoelectrons. This is detected by the photoelectric conversion elements of a total of 3 (channel) systems, which are the multipliers 23a and 23b and the photodiode 27.

In FIG. 1, the fluorescence from the sample 18 is dispersed by the dichroic mirror 20 according to its wavelength, and two kinds of fluorescent dyes are used. However, the dichroic mirror, the barrier filter, and the photomultiplier are further used. It goes without saying that fluorescence can be simultaneously detected from different fluorescent dyes by adding a tube.

Next, the structure of an electronic circuit for quantizing and storing the luminance signal obtained by the photomultiplier tubes 23a, 23b and the photodiode 27 will be described with reference to FIG. In the figure, the system of the luminance signal for the fluorescence received by the photomultiplier tube 23a is the first channel (CH1), the system of the luminance signal for the scattered light received by the photodiode 27 is the second channel (CH2), Photomultiplier tube 23
The system of the luminance signal for the fluorescence received in b is the third channel (CH3).

Photoelectric conversion circuit 3 comprising a photomultiplier tube 23a
1a, a photoelectric conversion circuit 31 including a photodiode 27
b, the light received by the photoelectric conversion circuit 31c including the photomultiplier tube 23b is converted by photoelectric conversion into a luminance signal of an analog value having a voltage corresponding to the luminance value.

The luminance signals obtained by the photoelectric conversion circuits 31a to 31c are amplified by the amplification circuits 32a to 32c individually for each channel with a constant amplification factor.
The luminance signal of the first channel amplified by the amplifier circuit 32a is directly fed to the data selector 34, and then the amplifier circuit 32b,
The luminance signals of the second and third channels amplified by 32c are respectively held by sample and hold circuits 33a and 33b and then sent to the data selector 34.

The sample and hold circuits 33a and 33b, the data selector 34, and the A / D converter 35 and the data selector 36, which will be described later, all operate in accordance with the sample clock sent from the sample clock generation circuit 38. The hold circuits 33a and 33b simultaneously sample and hold the luminance signals of the first channel from the amplifier circuit 32a and simultaneously hold the luminance signals of the second and third channels output from the amplifier circuits 32b and 32c. The luminance signals of the second and third channels which are sample-held are sequentially provided with an appropriate time lag until the new luminance signal of the first channel corresponding to the scanning position is output from the amplifier circuit 32a. The data is output to the data selector 34.

In the data selector 34, the amplifier circuit 3
The luminance signal of the first channel from 2a, the luminance signal of the second channel from the sample and hold circuit 33a, and the luminance signal of the third channel from the sample and hold circuit 33b are sequentially and cyclically selected by time division, and A It is sent to the / D converter 35.

The A / D converter 35 includes a data selector 34.
The luminance signals sequentially transmitted from the are synchronized with the sample clock from the sample clock generation circuit 38 are sequentially quantized with a predetermined number of quantization bits, and the obtained luminance signals of digital values are transmitted to the data selector 36.

Contrary to the data selector 34, the data selector 36 samples the luminance signals of the first to third channels of the digital value sent from the A / D converter 35 in a time division manner, and the sample clock generating circuit 38. Selectively distributes the brightness signal of the first channel to the memory 37a, the brightness signal of the second channel to the memory 37b, and the brightness signal of the third channel. The data is sent to the memory 37c and stored for each channel collectively.

Next, the signal flow in each of the first to third channels will be described with reference to FIG. In the figure, only one of the first to third channels is taken out and shown.

The light obtained from the sample 18 is converted into the photoelectric conversion circuit 3
1a (31b, 31c) converts into an analog luminance signal. The converted luminance signal is amplified by the amplifier circuit 32a (32b,
32c) to amplify (sample and hold circuits 33a, 3a
3b and) via the data selector 34, an A / D signal is generated by the sample clock from the sample clock generation circuit 38.
The converter 35 quantizes the luminance signal into a predetermined number of bits.

The quantized luminance signal is distributed through the data selector 36 and stored in the memory 37a (37b, 37b).
It is stored in c) for each channel. This memory 37a
The luminance signal stored in (37b, 37c) is directly sent to the image display unit 39 composed of, for example, a CRT and its drive circuit for image display as it is, or to obtain more detailed image data, The image is sent to the arithmetic circuit 40 and subjected to image processing by conversion processing such as digitization and graphing of the image, and then sent to the image display unit 39 for image display.

Accordingly, the sampling clock generation circuit 38, the amplification circuit 32a (32b, 32c), the A / D converter 35 and the memory 37a (37b, 37c) start and end the sampling, and the image display setting by the image display unit 39. , And the arithmetic setting by the arithmetic circuit 40 are
The scanning optical microscope apparatus is comprehensively controlled by a control command from a control circuit 41 which is the center of the system.

In the scanning optical microscope apparatus having the above structure, the process of photoelectrically converting the fluorescent light or scattered light emitted from the sample 18 and quantizing the fluorescent light or scattered light will be mainly described with reference to FIG.

In the figure, the optical signals for the first to third channels are converted into photoelectric conversion circuits 31a to 31c (photomultiplier tube 2).
3a, photodiode 27 and photomultiplier tube 23b)
Is converted into a luminance signal of an analog value, and its amplitude is amplified by the amplifier circuits 32a to 32c provided for each channel.

In the sample-hold circuit 33 provided on the second channel side and the sample-hold circuit 33b provided on the third channel side, the output of the amplifier circuit 32a is sent to the data selector 34 as it is. Second and third according to the quantization timing
Sample and hold the luminance signal of the channel.

The data selector 34 selects the luminance signal of the first channel sent directly from the amplifier circuit 32a at the timing of the input of the first sample clock to A / A.
After being sent to the D converter 35 and quantized, the luminance signal of the second channel held in the sample hold circuit 33a at the timing of the second sample clock input is selected and sent to the A / D converter 35. And quantize.

After that, the luminance signal of the third channel held in the sample hold circuit 33b is selected at the timing of inputting the third sample clock, and the A / D converter 35 is selected.
And quantize it.

The data selector 34 cyclically performs the above-mentioned operations to sequentially output the luminance signals of the first to third channels to the A / D converter 35 in a time division manner.

The luminance signals of the first to third channels of the digital value quantized by the A / D converter 35 are opposite to those of the data selector 34 due to the input of the sample clock from the sample clock generating circuit 38. Data selector 3 for selectively distributing luminance signals to
6, the memories 37a to 37c for each original channel
It is distributed to any one of the above and transmitted and stored.

Since one channel A / D converter 35 is used to quantize luminance signals for three channels in a time division manner,
It is not necessary to install D conversion equipment for each channel,
The cost can be reduced by reducing the circuit scale.

This is particularly remarkable as the number of quantization bits of the A / D converter increases. Further, since the luminance signal of each channel is quantized by the same single A / D converter 35, it is possible to design in consideration of characteristic error between channels when using a plurality of A / D converters. It becomes unnecessary.

[0044]

As described above, according to the present invention, A /
To reduce the circuit scale by reducing the number of D converters used as much as possible, and to quantize optical signals for multiple channels and store them for each channel without being affected by the characteristic error of the A / D converter. It is possible to provide an image processing circuit for a scanning microscope.

[Brief description of the drawings]

FIG. 1 is a diagram showing a mechanical and optical configuration of a scanning optical microscope apparatus according to an embodiment of the present invention.

FIG. 2 is a block diagram showing a configuration of a processing circuit for a plurality of channels of luminance signals according to the same embodiment.

FIG. 3 is a block diagram showing a flow of processing a 1-channel luminance signal according to the same embodiment;

FIG. 4 is a block diagram showing a configuration of a plurality of channels of luminance signal processing circuits used in a conventional scanning optical microscope apparatus.

[Explanation of symbols]

 1a, 1b, 31a to 31c ... Photoelectric conversion circuit 2a, 2b, 32a to 32c ... Amplification circuit 3a, 3b, 35 ... A / D converter 4, 38 ... Sample clock generation circuit 5a, 5b, 37a to 37c ... Memory 11 ... laser light source 12 ... spot projection lens 13, 20 ... dichroic mirror 14 ... galvano mirror 15 ... pupil projection lens 16 ... optical path switching mirror 17 ... objective lens 18 ... sample 19 ... scanning stage 21a, 21b ... barrier filter 22a, 22b ... collection Optical lens 23a, 23b ... Photomultiplier tube 24 ... Condenser lens 25 ... Beam splitter 26 ... Ring slit 27 ... Photodiode 28 ... Observation optical system 29 ... Transmission light source 33a, 33b ... Sample hold circuit 34, 36 ... Data selector 39 ... Image display unit 40 ... Arithmetic circuit 41 ... Control Circuit

Claims (1)

[Claims] 1. A scanning optical microscope that detects light from a sample obtained by two-dimensionally scanning a sample with a light beam, and processes the light to obtain a sample image. A photoelectric conversion means provided for each of a plurality of channels for photoelectric conversion, an amplification means for amplifying each analog value signal obtained by the photoelectric conversion means for each of a plurality of channels, and one of the signals amplified by these amplification means Holding means for respectively holding signals of other channels except one channel, and a signal for channels held by the holding means and signals for channels not held by the holding means are cyclically selected in a time division manner. Signal selecting means, converting means for converting an analog value signal sent via the first signal selecting means into a digital value, and this converting means for converting into a digital value Second signal selecting means for distributingly selecting the selected signal according to the channel, storage means for storing the signal of each channel distribution-selected by the second signal selecting means for each channel, and the above-mentioned holding Means, first signal selecting means, converting means and second
And a control means for performing timing control by supplying an operation clock to the signal selection means of.