JPH0972784A - Photometer for microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve a measurement with a high S/N ratio even if the luminous energy is very small by setting the dynamic range of the measurable luminous energy to a large value by automatically switching and selecting a count value in the case of very small luminous energy and the output value of an A/D conversion means in the other cases and outputting it as luminous energy information. SOLUTION: A numeric value comparison part 13 compares digital data outputted by an A/D converter 4 with a comparison value given from a comparison value setting part 14. A data selection part 7 selects digital data outputted by the converter 4 when the output signal from the converter 4 is larger the comparison value and the count value of a pulse coefficient part 6 as digital data when the output signal is smaller and then outputs it to a memory 8. In this case, since the comparison part 13 performs a comparison operation using the output of the converter 4 which is a digital value, an accurate and speedy operation can be made without generating a comparison error and an output delay in analog-like comparison operation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a photometric device for a microscope using a photoelectric conversion element having a photoelectron amplification function such as a photomultiplier tube.

[0002]

2. Description of the Related Art A photometric microscope irradiates an observation sample with illumination light from a light source, whereby transmitted light transmitted through the observation sample,
Fluorescent light generated from the sample by irradiating the reflected light or point-shaped light is imaged through an optical system that includes an objective lens, and this is detected by a photodetector placed at the imaging position. Then, the information on the amount of light is obtained.

Further, the photometric microscope uses a sample on the X-axis and Y-axis.
An XY scanning system that mechanically moves in a two-dimensional plane by moving in the axial direction and an optical system that scans the illumination light are adopted, and CR is synchronized with these XY scanning.
A scanning optical microscope is also included, which allows a sample to be observed and analyzed as an image by displaying bright points of the signal of the density information corresponding to XY scanning by a display device such as a T display.

In general, these photometric microscopes convert transmitted light, reflected light, and fluorescence from an illuminated sample into an electric signal by a photoelectric converter, further quantizing the electric signal, and then storing this as an image or temporal change data. A photoelectric signal processing circuit for

In particular, in order to detect fluorescence from which only a weak light amount is obtained or light from a confocal optical system frequently used in a scanning optical microscope, a photoelectric converter, a photomultiplier tube, or a multichannel is used as a photoelectric converter. A plate, avalanche photodiode, or other highly sensitive one having a photoelectron amplification effect is often used.

When using a high-sensitivity one having this type of photoelectron amplification effect, when using an A / D converter as a quantizing circuit for an analog signal obtained from a photoelectric converter, the dynamic range is increased. If a logarithmic amplifier is used in front of the A / D converter as in Japanese Patent Laid-Open No. 58-139036, the amount of light to be measured is extremely small. When using a photon counting circuit that counts the pulse of a signal, an optimal circuit configuration is selected and used according to the amount of light to be measured.

[0007]

Particularly, the measuring object of a photometric microscope typified by a scanning optical microscope is very wide in a two-dimensional plane from a bright portion having a relatively large amount of light to a dark portion having a relatively small amount of light. A wide range of light needs to be measured.

However, it is difficult for a photometry circuit using a general A / D converter to measure a portion with a small light amount without lowering the S / N ratio, and photometry using a photon counting circuit. In the circuit, the part with low light intensity has a high S / N ratio.
Although it can be measured by a ratio, there is a problem that measurement becomes impossible when the amount of light exceeds a certain amount.

The present invention has been made in view of the above circumstances, and a microscope capable of performing measurement with a high S / N ratio even with a minute light amount while setting a large dynamic range of the measurable light amount. It is to provide a photometric device for use.

[0010]

According to the first aspect of the present invention,
The light obtained by illuminating the observation sample with the illumination light from the light source,
In a photometric device for an optical microscope, which forms an image through an optical system including an objective lens and detects the amount of light with a photodetector to obtain light amount information, the detection signal from the photodetector is amplified to digital from an analog value. A / D converting means for converting into a value, counting means for amplifying the pulse component of the detection signal from the photodetector to count the number of photons, and the A / D according to the detection signal level of the photodetector. A selection means for selecting one of the output of the conversion means and the count value of the counting means as measurement information is provided.

As a result, according to the invention of claim 1,
When the light quantity is very small, the count value of the counting means is automatically switched and selected in other cases, and the output value of the A / D conversion means is automatically selected and output as the light quantity information.
It is possible to perform measurement with a high S / N ratio even with a minute light amount while setting a large dynamic range of the measurable light amount.

According to a second aspect of the present invention, in the first aspect of the present invention, the selection means counts the output of the A / D conversion means and the count means according to the output level of the A / D conversion means. I am trying to select either one of the values.

According to a third aspect of the present invention, in the first aspect of the present invention, the selecting means selects either the output of the A / D converting means or the count value of the counting means according to the count value of the counting means. I'm trying to choose one or the other.

As a result, according to the invention as claimed in claim 2 or claim 3, since the comparison operation is performed using the output of the A / D conversion means which is a digital value, the comparison operation for analog comparison is performed. An accurate and high-speed operation can be performed without a fear of causing a comparison error or an output delay which is a concern in a device.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION

(First Embodiment) FIG. 1 illustrates a configuration centering on an optical system of a scanning optical microscope according to a first embodiment of the present invention, and 21 is a laser light source as a light source. . The illumination light from the laser beam emitted from the laser light source 21 passes through the beam expander 22 and the wavelength filter 23, and is deflected by the optical path splitting means 24 including a beam splitter as the optical path splitting means 24 and a dichroic mirror. Are guided to galvanometer mirrors 25 and 26, where they are deflected in the X-axis direction and the Y-axis direction for two-dimensional scanning.

This deflected illumination light is used for the pupil projection lens 2
7, the light enters the objective lens 29 through the prism 28, and the observation sample 30 is two-dimensionally scanned. The transmitted light of the observation sample 30 is condensed by a lens 31, reflected by a mirror 32, photoelectrically converted by a photodetector 34 via a lens 33, digitized by an A / D converter (not shown), and then X -It is stored in the frame memory 35 at the coordinate position corresponding to the Y scanning position.

At least one of reflected light and fluorescence obtained by irradiation of the observation sample 30 is guided to the half mirror 24 along the same path as the irradiation light, and passes through the half mirror 24 and the condenser lens 36. Are reflected by the dichroic mirrors 37 and 42 disposed on the optical path.

The light reflected by the dichroic mirror 37 enters the photodetector 1 through the absorption filter 38 and the pinhole plate 39 and is converted into an electric signal corresponding to the amount of light.

The photodetector 1 uses, for example, a photomultiplier tube, a multi-channel plate, an avalanche photodiode or the like having a high sensitivity having a photoelectron amplifying action, and an electric signal as a detection signal thereof is used as a photometric circuit 41. And output as light amount information.

Almost similarly, the light reflected by the dichroic mirror 42 enters the photodetector 1'through the dichroic filter 43, the absorption filter 44, and the pinhole plate 45, and is converted into an electric signal corresponding to the amount of light. To be done.

This photodetector 1'is also the above photodetector 1,
Similarly to 1 ', a highly sensitive one having a photoelectron amplification function such as a photomultiplier tube, a multi-channel plate, or an avalanche photodiode is used. Is output as.

Next, referring to FIG. 2, the photodetector 1 (1 ')
The detailed configuration of the photometric circuit 41 (40) at the subsequent stage will be described. In the figure, an electric signal having a current value corresponding to the light amount obtained by the photodetector 1 (1 ') is first converted into a voltage value corresponding to the light amount by the current-voltage converter 2 in the photometric circuit 41 (40). The signal is converted into a signal having the signal amplification unit 3
And the pulse amplification unit 5.

The signal amplification section 3 amplifies the input voltage signal with a preset appropriate amplification factor, and outputs the amplified voltage signal to the A / D converter 4 and the voltage comparison section 11. The A / D converter 4 converts the analog-valued voltage signal from the signal amplification section 3 into a digital value with a predetermined number of quantization bits in synchronization with the sampling clock from the clock generation section 9, and sends it to the data selection section 7. Output.

The voltage comparison unit 11 is composed of a comparator such as a high speed comparator, and clocks the voltage signal from the signal amplification unit 3 and the reference voltage as a threshold value set by the reference voltage setting unit 12. The comparison is performed in synchronization with the clock from the unit 9, and the comparison result is output to the data selection unit 7.

On the other hand, the pulse amplifier 5 amplifies only the pulse component (AC component) of the voltage signal from the current-voltage converter 2 and outputs it to the pulse counter 6. The pulse counter 6 is
It has a function of setting a threshold value for pulse detection, and counts the number of shaping pulses in the pulse waveform from the pulse amplification unit 5 for each cycle of the sampling clock input from the clock generation unit 9, and The count value is output to the data selection unit 7 as needed.

The data selection section 7 synchronizes with the sampling clock from the clock generation section 9 and the voltage comparison section 1 described above.
When the output signal from the signal amplification unit 3 is larger than the reference voltage based on the comparison result from 1, the digital data output from the A / D converter 4 is set to the reference voltage when the output signal from the signal amplification unit 3 is set to the reference voltage. When it is smaller than the above, the count value of the pulse counting unit 6 is selected as digital data, and as the measurement result, the digital data of the selected light amount information and any output of the A / D converter 4 and the pulse counting unit 6 are selected. The selection information indicating whether or not is output to the memory 8.

The memory 8 is composed of, for example, a frame memory having a capacity capable of storing at least one frame of image, and the digital data from the data selecting section 7 is
The data is stored according to the designated address input from the address control unit 10. The address controller 10 synchronizes with the sampling clock input from the clock generator 9 and
An address position corresponding to the one-to-one correspondence with the position of the observation sample 30 currently being sampled is updated and stored, and the address position is given to the memory 8 as a designated address.

However, the digital data of the measurement result stored in the memory 8 is read out by the data processing unit 17 and subjected to various image analysis processing to obtain desired image information, and then the CRT or LCD panel is used. Is displayed on the display unit 18 that has been displayed.

The data processing section 17 and the reference voltage setting section 12 are both controlled by a system control section 16 which is, for example, a personal computer.

That is, the system control unit 16 allows the reference voltage setting unit 12 to arbitrarily set the above-mentioned reference value, so that the measurement image displayed on the display unit 18 can be observed and corresponding to the contents. The reference value can be adjusted.

On the other hand, the system control unit 16 instructs the data processing unit 17 on the method of image analysis processing, so that the image information of the observation sample 30 to be displayed on the display unit 18 can be arbitrarily selected. .

In the configuration as described above, the voltage comparison unit 1
1 is an amplified voltage signal output from the signal amplification unit 3, that is, a signal corresponding to the amount of light incident on the photodetector 1 (1 ') is compared with a reference value from the reference voltage setting unit 12, and the comparison result indicates data. The selecting unit 7 switches and selects one of the output of the A / D converter 4 and the output of the pulse counting unit 6 for each clock from the clock generating unit 9 and stores the quantized light amount information in the memory 8. Therefore, the light amount can be measured in a very wide dynamic range from weak light that cannot be measured without counting photons to relatively bright light.

(Second Embodiment) FIG. 3 shows a second embodiment of the present invention.
The photodetector 1 (1 ') used in the optical system of the scanning optical microscope according to the embodiment of the present invention and the photometry circuit 41 (4) at the subsequent stage.
The detailed configuration of (0) is illustrated, and since it is basically the same as that shown in FIG. 2, the same portions are denoted by the same reference numerals and the description thereof will be omitted.

Therefore, the digital data obtained by the A / D converter 4 is not limited to the data selection section 7 but the numerical comparison section 13 as well.
Is also output to The numerical comparison section 13 outputs from the clock generation section 9 the comparison value sent from the comparison value setting section 14 which is variably set by the system control section 16 and the digital data output from the A / D converter 4. The data are compared with each other in synchronization with the sampling clock of and the comparison result is output to the data selection unit 7.

With the above structure, A /
The digital comparison unit 13 uses the digital data output from the D converter 4 to compare it with the comparison value provided from the comparison value setting unit 14, and the data selection unit 7 determines A / A according to the comparison result.
When the output signal from the D converter 4 is larger than the comparison value, the digital data output from the A / D converter 4 is changed to A / D.
When the output signal from the converter 4 is smaller than the comparison value, the count value of the pulse counter 6 is selected as digital data and output to the memory 8.

In this case, since the numerical comparison section 13 performs the comparison operation using the output of the A / D converter 4 which is a digital value, a comparator such as a high speed comparator which performs an analog comparison operation. Compared with the configured voltage comparison unit 11 of FIG. 2, it is possible to perform an accurate and high-speed operation without fear of causing a comparison error or an output delay.

(Third Embodiment) FIG. 4 shows a third embodiment of the present invention.
The photodetector 1 (1 ') used in the optical system of the scanning optical microscope according to the embodiment of the present invention and the photometry circuit 41 (4) at the subsequent stage.
The detailed configuration of (0) is illustrated, and since it is basically the same as that shown in FIGS. 2 and 3, the same portions are denoted by the same reference numerals and the description thereof will be omitted.

Thus, the count value obtained by the pulse counting section 6 is output as digital data to the numerical value comparing section 13 as well as the data selecting section 7. This numerical comparison unit 13
The clock generator 9 compares the comparison value sent from the comparison value setting unit 14 variably set by the system control unit 16 and the digital data output from the pulse counting unit 6.
The data is compared with each other in synchronization with the sampling clock from the above and the comparison result is output to the data selecting section 7.

With the above-mentioned configuration, the numerical comparison section 13 compares the digital data output from the pulse counting section 6 with the comparison value given from the comparison value setting section 14, and the comparison result is obtained. When the output signal from the pulse counting unit 6 is larger than the comparison value in the data selecting unit 7, the digital data output from the A / D converter 4 is smaller than the comparison value. At this time, the count value of the pulse counter 6 is selected as digital data and output to the memory 8.

In this case, since the numerical value comparing section 13 performs the comparing operation using the count value of the pulse counting section 6 which is a digital value, an analog comparison is performed as in the case of the second embodiment. For example, the voltage comparison unit 1 of FIG. 2 configured to include a comparator such as a high speed comparator that operates.
Compared with 1, it is possible to perform accurate and high-speed operation without fear of causing a comparison error or output delay.

(Fourth Embodiment) FIG. 5 shows a fourth embodiment of the present invention.
The photodetector 1 (1 ') used in the optical system of the scanning optical microscope according to the embodiment of the present invention and the photometry circuit 41 (4) at the subsequent stage.
The detailed configuration of (0) is illustrated, and since it is basically the same as that shown in FIGS. 2 to 4, the same parts are denoted by the same reference numerals and the description thereof will be omitted.

The voltage signal obtained by amplifying only the pulse component output from the pulse amplifier 5 is sent to the pulse width detector 15 as well as the pulse counter 6. The pulse width detection unit 15 measures the pulse width in the voltage signal and sends the measurement result to the numerical comparison unit 13.

On the other hand, the numerical comparison section 13 has a comparison value setting section 14 which is variably set by the system control section 16.
The comparison value sent from the pulse width detection unit 15 and the measured value of the pulse width from the pulse width detection unit 15 are compared in synchronization with the sampling clock from the clock generation unit 9, and the comparison result is output to the data selection unit 7. It is a thing.

With the above configuration, the amount of light incident on the photodetector 1 (1 ') is small, and the voltage signal output from the current-voltage converter 2 has a waveform as shown in FIG. 6A, for example. When the pulse width is 1, the pulse amplification unit 5 sends a voltage signal whose pulse width is amplified with reference to the pulse detection threshold in FIG. 5A to the pulse counting unit 6 and the pulse width detection unit 15, and the pulse width detection unit 15 Measures the pulse width and sequentially outputs the measured values to the numerical comparison unit 13.

The numerical comparison section 13 compares the comparison value sent from the comparison value setting section 14 with the measured pulse width value from the pulse width detection section 15 in synchronization with the sampling clock from the clock generation section 9. The comparison result is sent to the data selection unit 7, and the data selection unit 7 uses the comparison result to detect the photodetector 1.
Recognizing that the amount of light incident on (1 ') is small, A /
Instead of the digital data output from the D converter 4, the count value of the pulse counting unit 6 is switched and selected as digital data and output to the memory 8.

On the contrary, when the amount of light incident on the photodetector 1 (1 ') is large and the voltage signal output from the current-voltage converter 2 has a waveform as shown in FIG. 6B, for example, the pulse amplifier. When the voltage signal amplified by 5 is sent to the pulse counter 6 and the pulse width detector 15, the pulse width detector 15 measures the pulse width with reference to the pulse detection threshold in FIG. Is output to the numerical comparison unit 13.

In this case, in FIG. 6 (a), four photons are counted in one clock, while in FIG. 6 (b), the amount of light is large and many photons are incident on the photodetector. However, the number of photons to be counted becomes three.

Therefore, when the pulse width detection unit 15 measures the pulse width of the portion exceeding the pulse detection threshold value and outputs the measured value to the numerical comparison unit 13, the numerical comparison unit 1
Reference numeral 3 denotes a comparison value sent from the comparison value setting unit 14 and a pulse width measurement value from the pulse width detection unit 15 in synchronization with a sampling clock from the clock generation unit 9 to compare the comparison result with a data selection unit. Send to 7.

The data selecting section 7 recognizes from the comparison result that the light quantity incident on the photodetector 1 (1 ') is large and the pulses of the voltage signal of the light quantity information are overlapped, and the pulse counting section 6 Instead of the output count value, the digital data output from the A / D converter 4 is switched and selected and output to the memory 8.

In this way, the pulse width detection section 15 measures the pulse width of the output of the pulse amplification section 5 located before the pulse counting section 6, and the numerical comparison section 13 determines whether or not the pulses overlap based on the measured value. Even if the comparison operation is performed, there is no possibility of causing a comparison error or an output delay as compared with the voltage comparison unit 11 of FIG. 2 configured by a comparator such as a high speed comparator which performs an analog comparison operation. Therefore, it becomes possible to perform accurate and high-speed operation.

The first to fourth embodiments described above can be combined with each other to set more strict selection conditions. Although the above description has been given based on the respective embodiments, the present invention includes the following inventions.

(1) The light obtained by irradiating the observation sample with the illumination light from the light source is imaged through the optical system including the objective lens, and the light quantity is detected by the photodetector to obtain the light quantity information. In an optical microscope photometric device, A / D conversion means for amplifying a detection signal from the photodetector to convert it from an analog value to a digital value, and a pulse component of the detection signal from the photodetector for amplifying a photon Counting means for counting the number and the A / D depending on the detection signal level of the photodetector
A photometric device for a microscope, comprising: selecting means for selecting one of the output of the converting means and the count value of the counting means as measurement information.

By doing so, the count value of the counting means is used when the light quantity is very small, and the A / D value is used in other cases.
Since the output value of the conversion means is automatically switched and selected and output as the light amount information, a high S / N ratio is measured even with a small light amount while setting a large dynamic range of the measurable light amount. be able to.

(2) The selecting means is characterized by selecting one of the output of the A / D converting means and the count value of the counting means according to the output level of the A / D converting means ( A photometric device for a microscope according to the item 1).

(3) The selecting means selects one of the output of the A / D converting means and the count value of the counting means according to the count value of the counting means. The photometric device for a microscope described.

(4) The selection means selects one of the output of the A / D conversion means and the count value of the counting means according to the pulse width of the pulse signal input to the count. The photometric device for a microscope according to item (1).

According to the above items (2) to (4),
In addition to the above item (1), since the comparison operation is performed using a digital value, there is no fear of causing a comparison error or an output delay, which is a concern in a comparator performing an analog comparison operation. A high speed operation can be performed.

[0058]

As described above in detail, according to the present invention, a photometric device for a microscope capable of performing measurement with a high S / N ratio even with a minute light amount while setting a large dynamic range of the measurable light amount. Can be provided.

[Brief description of drawings]

FIG. 1 is a diagram illustrating a configuration centered on an optical system of a scanning optical microscope according to a first embodiment of the present invention.

FIG. 2 is a block diagram mainly showing a detailed circuit configuration in the photometric circuit of FIG.

FIG. 3 is a block diagram mainly showing a detailed circuit configuration in a photometry circuit according to a second embodiment of the present invention.

FIG. 4 is a block diagram mainly showing a detailed circuit configuration in a photometry circuit according to a third embodiment of the present invention.

FIG. 5 is a block diagram mainly showing a detailed circuit configuration in a photometry circuit according to a fourth embodiment of the present invention.

FIG. 6 is a diagram showing an example of operation waveforms according to the same embodiment.

[Explanation of symbols]

 1, 1 ', 34 ... Photodetector 2 ... Current / voltage conversion unit 3 ... Signal amplification unit 4 ... A / D converter 5 ... Pulse amplification unit 6 ... Pulse counting unit 7 ... Data selection unit 8 ... Memory 9 ... Clock generation Part 10 ... Address control part 11 ... Voltage comparison part 12 ... Reference voltage setting part 13 ... Numerical value comparison part 14 ... Comparison value setting part 15 ... Pulse width detection part 16 ... System control part 17 ... Data processing part 18 ... Display part 21 ... Laser light source 22 ... Beam expander 23 ... Wavelength filter 24 ... Optical path splitting means 25, 26 ... Deflection means 27 ... Pupil projection lens 28 ... Prism 29 ... Objective lens 30 ... Observation sample 31, 33 ... Lens 32 ... Mirror 35 ... Frame memory 36 ... Condensing lens 37, 42 ... Dichroic mirror 38, 44 ... Absorption filter 39, 45 ... Pinhole plate 40, 41 ... Photometric circuit 43 ... Die Roy click filter

Claims (3)

[Claims] 1. An optical system in which light obtained by irradiating an observation sample with illumination light from a light source is imaged through an optical system including an objective lens, and the light amount is detected by a photodetector to obtain light amount information. In a photometric device for a microscope, A / D conversion means for amplifying a detection signal from the photodetector to convert it from an analog value to a digital value, and a pulse component of the detection signal from the photodetector to amplify the number of photons. Counting means and a selecting means for selecting one of the output of the A / D converting means and the count value of the counting means as measurement information according to the detection signal level of the photodetector. A photometric device for microscopes characterized by the above. 2. The selecting means selects either one of the output of the A / D converting means and the count value of the counting means in accordance with the output level of the A / D converting means. 1. A photometric device for a microscope according to 1. 3. The selection means selects one of the output of the A / D conversion means and the count value of the counting means according to the count value of the counting means. Photometric device for microscopes.