JP2003098086A - Light intensity measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a light intensity measuring device capable of obtaining an image of high quality having an approximately constant S/N regardless of the light intensity, and securing a high image forming speed. SOLUTION: The light obtained by applying the irradiation light from a light source to an observation sample, or the light emitted from the observation sample is detected by a light-sensitive detector 1 as photons, the number of detected photons is counted by a pulse counting part 3, a sampling time is counted by a clock counting part 4 until the counted number reaches the set number of photons set in a pulse number setting part 8 wherein the predetermined number of photons is set in advance, and the information on brightness of the observation sample is determined in a data processing part 9 on the basis of the set number of photons of the pulse number setting part 8 and the sampling time counted by the clock counting part 4.

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 use in a scanning optical microscope for acquiring a fluorescent image of biological cells.

[0002]

2. Description of the Related Art Conventionally, in a scanning optical microscope, in order to quantitatively measure a fluorescent substance (or fluorescent molecule) in a sample, a part of the sample to be observed is irradiated with a laser as excitation light for a certain period of time. A light quantity photometric device is used that measures fluorescence emitted from a fluorescent substance for a certain period of time.

However, in such a light quantity photometric device, measurement is performed for the same amount of time in a place where the fluorescence is small and a place where the fluorescence is large. Therefore, in a place where the fluorescence is small, no photon is incident within a certain period of time. The amount of light cannot be measured at all. Therefore, in order to solve such a problem, it is conceivable to lengthen the measurement time, but in this method, the laser is excessively irradiated in the portion where much fluorescence is generated, which causes fading. .

In addition, in the past, for example, Japanese Patent Laid-Open No. 9-7
As disclosed in Japanese Patent No. 2784, in a photometric device for a microscope using a photoelectron conversion element having a photomultiplier function such as a photomultiplier tube, a photon counting method circuit and A A circuit using a D / D converter is appropriately switched.
As disclosed in Japanese Laid-Open Patent Publication No. 60380, a charge storage type image sensor circuit is used, and the current flowing when light is incident on the image sensor is stored in a capacitor, and the time until a constant charge amount is obtained is defined as brightness. As disclosed in Japanese Patent Application Laid-Open No. 6-313840, the photocurrent generated by the light receiving element is stored in a capacitor, a certain amount of charge is stored, and then discharged at a constant current.
It is also considered that the discharge time is measured and treated as brightness.

[0005]

However, Japanese Patent Laid-Open No. 9-
Japanese Patent Laid-Open No. 72784 discloses a method of measuring weak light using a photon counting method in order to widen the dynamic range. When applied to a scanning optical microscope for living organisms, if the scanning speed is slowed, Since the irradiation time becomes longer, fading of biological specimens becomes faster, and conversely, if the scanning speed is increased, it will be difficult to image a part with a small amount of light because a sufficient amount of light cannot be obtained. Occurs.

Further, Japanese Patent Laid-Open No. 2-60380 discloses a charge storage time measuring method in which a current flowing when light is incident on an image sensor is stored in a capacitor, and the storage time is measured. The one disclosed in Kaihei 6-313840 is a charge discharge time measuring method in which charges once stored in a capacitor are discharged and the discharge time is measured. Since neither of these can measure light at the same time as scanning, image acquisition is performed. There is a problem that it is difficult to apply to a scanning optical microscope that requires high speed.

The present invention has been made in view of the above circumstances, and it is possible to obtain a good image in which the S / N is constant in a place with a large amount of light and a place with a small amount of light, and to secure a quick image acquisition speed. It is an object of the present invention to provide a light quantity measuring device that can be used.

[0008]

The invention according to claim 1 is
Light detection means for detecting, as photons, light obtained by irradiating an observation sample with illumination light from a light source or light emitted by the observation sample, light amount setting means for setting a light amount corresponding to a predetermined number of photons in advance, and the light Time measuring means for measuring the time until the light quantity corresponding to the number of photons detected by the detecting means reaches the light quantity set in the light quantity setting means, a predetermined light quantity set in the light quantity setting means and the time measuring means And a brightness information calculating means for calculating the brightness information of the observation sample according to the time measured in step 1.

According to a second aspect of the present invention, light detection means for detecting, as photons, light obtained by illuminating the observation sample with illumination light from the light source or light emitted by the observation sample, and the light detection means detects the light. Photon counting means for counting the number of photons, photon number setting means for setting a predetermined number of photons in advance, and sampling until the photon count value of the photon counting means reaches the set number of photons set in the photon number setting means. It is characterized by comprising time measuring means for measuring time, and brightness information calculating means for calculating the brightness information of the observation sample based on the set photon number of the photon number setting means and the sampling time measured by the time measuring means. I am trying.

According to a third aspect of the present invention, light detection means for detecting, as photons, light obtained by irradiating the observation sample with illumination light from a light source or light emitted by the observation sample, and the light detection means detect the light. Integrating means for integrating the output corresponding to the number of photons, threshold setting means for setting a predetermined threshold value in advance, and the integral value of the integrating means for the threshold value set for the threshold setting means. A time measuring means for measuring the sampling time until reaching, and a brightness information calculating means for calculating the brightness information of the observation sample by the threshold of the threshold setting means and the sampling time measured by the time measuring means. It is characterized by having.

As a result, according to the present invention, when measuring a portion of the observation sample having a small amount of light, it takes a long time to be measured by the time measuring means, and conversely, when measuring a portion having a large amount of light. The time measured by the time measuring means is shortened, and the light irradiation time can be shortened. This allows
Since the same number of photons can be measured at a place with a large amount of light and a place with a small amount of light, a good image with a substantially constant S / N can be obtained. Further, since photometry can be performed at the same time as scanning, optimal light amount measurement can be performed even in a scanning optical microscope that requires a rapid image acquisition speed.

Further, by using the charge storage method, accurate measurement can be performed even when the number of photons per unit time is large, which cannot be accurately measured by the method of measuring the number of photons.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

(First Embodiment) FIG. 1 shows a first embodiment of the present invention.
2 shows a schematic configuration centering on an optical system of a scanning optical microscope to which the light quantity measuring device according to the embodiment is applied.

In the figure, reference numeral 21 denotes a laser light source as a light source. Illumination light by a laser beam emitted from the laser light source 21 is composed of a beam splitter, a dichroic mirror, etc. after passing through a beam expander 22 and a wavelength filter 23. It is guided to the galvanometer mirrors 25 and 26 as the deflecting means by the optical path splitting means 24, and is deflected in the X-axis direction and the Y-axis direction for two-dimensional scanning.

The illumination light deflected by the galvanometer mirrors 25 and 26 is used as a pupil projection lens 27 and a prism 28.
It is incident on the objective lens 29 via the, and the observation sample 30 is two-dimensionally scanned. The transmitted light of the observation sample 30 is condensed by the lens 31, reflected by the mirror 32, photoelectrically converted by the photodetector 34 via the lens 33, and is transmitted to the frame memory at the coordinate position corresponding to the XY scanning position. The light amount information is stored in 35.

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

The light reflected by the dichroic mirror 37 is incident on the photodetector 1 via the absorption filter 38 and the pinhole plate 39, where it is converted into an electric signal corresponding to the amount of incident light. This photodetector 1 uses, for example, a photomultiplier tube, an avalanche photodiode or the like having a high sensitivity having a photoelectron amplifying action, and an electric signal which is a detection signal thereof is sent to the photometric circuit 41 to obtain light quantity information. Is output.

The light transmitted through the dichroic mirror 37 and reflected by the mirror 42 enters the photodetector 1'through the dichroic filter 43, the absorption filter 44, and the pinhole plate 45, and corresponds to the number of photons. Is converted into an electric signal. Like the photodetector 1, this photodetector 1'also uses a highly sensitive one having a photoelectron amplifying action such as a photomultiplier tube or an avalanche photodiode, and the electric signal as the detection signal is a photometric circuit. It is sent to 40 and is output as light amount information.

The light amount information output from the photometric circuit 41 (40) is sent to the main control unit 50. Main control unit 50
Is to obtain the brightness information of the observation sample based on the light amount information from the photometric circuit 41 (40). The brightness information is given to the light irradiation control unit 51 to control the laser light source 21 and the galvanometer mirrors 25, 26. It has become.

FIG. 2 shows a schematic structure of the photometric circuit 41 (40), the main controller 50 and the light irradiation controller 51. In this case, the number of photons obtained by the photodetector 1 (1 ') is shown. In the photometric circuit 41 (40), the current pulse corresponding to the current pulse is first obtained by the current-voltage conversion unit 2 corresponding to the number of photons. The pulse counting unit 3 counts the pulses until reaching the set pulse number set in the pulse number setting unit 8 as the light amount setting means. When the first pulse is counted and the pulse number reaches the set value. At that time, a signal indicating the start and end of pulse counting is sent to the clock counting unit 4, the address control unit 6, and the system control unit 11.

The clock counting section 4 constitutes a time measuring means, counts the clocks generated by the clock generating section 5, and confirms that the pulse count value from the pulse counting section 3 has reached the set pulse number. From the signal shown, the number of clocks used for pulse counting is output to the memory 7 as digital data. The memory 7 is composed of, for example, a frame memory having a capacity capable of storing at least one frame image, and stores the digital data from the clock counting unit 4 in accordance with a designated address input from the address control unit 6.

The address control unit 6 synchronizes with the signal indicating the end of pulse counting, which is input from the pulse counting unit 3,
An address position corresponding to the position of the observation sample currently being sampled is updated and stored, and the address position is given to the memory as a designated address. The digital data of the measurement result stored in the memory 7 is read by the data processing unit 9 which constitutes the brightness information calculating means in the main control unit 50 and is used as the brightness information (the set number of photons).
Calculate / (number of clocks). After that, after various image analysis processing is performed to obtain desired image information, CRT or L
It is displayed on the display unit 10 using a CD panel.

Data processing unit 9 and pulse number setting unit 8
Are all controlled by the system control unit 11 which is composed of, for example, a personal computer. That is, the system control unit 11 makes the pulse number setting unit 8
By setting the number of set pulses to be arbitrarily set, the measurement image displayed on the display unit 10 is observed,
The set pulse number can be adjusted according to the content. On the other hand, when the system control unit 11 instructs the data processing unit 9 on the image analysis processing method, the display unit 10
The image information of the observation sample 30 to be displayed can be selected arbitrarily. Further, the system control unit 11 controls the irradiation light in synchronization with the start or end of pulse counting, so that the galvanometer mirror control unit 12 and the light emission control of the light irradiation control unit 51 are controlled by a signal indicating the end of pulse counting. A control signal is output to the unit 13 (for example, a shutter control unit or an acousto-optic device control unit) so that light is not emitted while the galvanometer mirrors 25 and 26 are driving, and the galvanometer mirror 25 emits light again when the galvanometer mirrors 25 and 26 are stationary. 26
And the light emission of the laser light source 21 are controlled.

Next, the operation of the embodiment thus constructed will be described with reference to the flow chart shown in FIG.

In this case, first, the system control unit 11 sets the set pulse number in the pulse number setting unit 8 (step 301), and then the clock count value of the clock counting unit 4 and the pulse counter of the pulse counting unit 3 are set. Initialize the numbers
At steps 302 and 303, the observation sample 30 is irradiated with the laser light of the laser light source 21 by the light emission controller 13 (step 304).

Next, it is judged whether or not the first photon comes from the photodetector 1 (1 ') (step 305), and when the first photon comes, the current pulse corresponding to the number of photons is pulse counted. The counting is performed by the unit 3 (step 306). Further, the clock counting unit 4 counts the number of clocks spent until the photon arrives (step 307).

It is judged whether the number of pulses counted by the pulse counting section 3 has reached the set number of pulses (step 308). If not, the steps 306 to 308 are repeated.

After that, when the number of counting pulses of the pulse counting unit 3 reaches the set number of pulses, the light emission control unit 13 prevents the laser light of the laser light source 21 from irradiating the observation sample 30 (step 309), In response to a signal indicating that the pulse count value of the counting unit 3 has reached the set number of pulses, the clock count value used for pulse counting up to now is output as digital data to the memory 7 and input from the address control unit 6. The data is stored in the memory 7 according to the designated address (step 310).

Next, the light emission control unit 13 instructs the galvanometer mirrors 25 and 26 to the galvanometer mirror control unit 12.
Directs the laser light of the laser light source 21 to irradiate the next measurement position on the observation sample 30, and further instructs the address controller 6 to specify the address on the memory 7 corresponding to the next measurement position. Instruct (steps 311, 31)
2). Then, the digital data of the measurement result stored in the memory 7 is read to the data processing unit 9 of the main control unit 50, and (the number of set photons) / (the number of clocks) is calculated as the brightness information (step 313). The brightness information is displayed on the display unit 10 (step 314). Then, it is judged whether or not the image acquisition is to be ended (step 315), and if it is not ended, the operations of steps 302 to 315 described above are repeated.

According to such a configuration, the photodetector 1
The current pulse corresponding to the photon from (1 ') is counted, and the time until this pulse reaches the set pulse number is measured, and the light irradiation to the observation sample 30 is controlled in synchronization with it. Therefore, when the light amount of the observation sample 30 is being measured, light irradiation is performed until the set number of photons is counted, while conversely, when measuring the part having a large amount of light, the set photon number is obtained. The light irradiation time can be shortened by shortening the time until. As a result, the same number of photons is measured at a place with a large amount of light and a place with a small amount of light. Therefore, the image displayed on the display unit 10 is a good image with a constant S / N when noise other than quantum noise is ignored. In addition, since photometry can be performed simultaneously with scanning, optimal light amount measurement can be performed even in a scanning optical microscope that requires a rapid image acquisition speed.

Further, the light emission control unit 13 is connected to the pulse counting unit 3
By controlling with, for example, when measuring multiple lasers and channels at the same time, it is possible to control so as not to irradiate the laser of the channel that has reached the set number of photons earlier than other channels. Irradiation of laser is eliminated, and fading of the observation sample 30 can be prevented.

Further, since the light is prevented from hitting the observation sample 30 during the operation of the galvanometer mirror, it is possible to carry out the measurement while suppressing the fading of the observation sample 30.

(Second Embodiment) In the second embodiment, a schematic configuration diagram of a scanning optical microscope to which the light quantity measuring device described in the first embodiment is applied, a photometry circuit 41 (4)
0), the schematic configuration diagrams of the main control unit 50 and the light irradiation control unit 51 are the same as those in FIGS. 1 and 2, and thus these drawings are incorporated herein.

In this case, the system control unit 11 of the main control unit 50 receives the light emission signal synchronized with the light emission from the light emission control unit 13 to the pulse counting unit 3 in synchronization with the light emission signal or It is designed to give an instruction to perform pulse counting at a fixed timing.

In the second embodiment as described above, the flowchart shown in FIG. 4 is executed. In this case, steps 401 to 403 correspond to step 301 shown in FIG. 3 described above.
~ 303. Then, the light emission signal synchronized with the light emission of the light emission control unit 13 is sent to the pulse counting unit 3 through the system control unit 11, and it is determined whether or not it is in the countable timing that matches the light emission signal. (Step 4
04), at this timing, the current pulse corresponding to the number of photons is counted by the pulse counter 3 (step 405). Further, the clock counting unit 4 counts the number of clocks spent until the arrival of this photon (step 406).

The following steps 407 to 413 are
Step 3 except step 309 shown in FIG. 3 described above
It is the same as 08-315.

With this configuration, the pulse counter 3
The operation and non-operation can be matched with the light emission, non-emission (non-emission, emission) by the light emission control unit 13 or a certain timing related thereto. Therefore, for example, the laser light source 2
When 1 is a pulsed laser, the photon counting can be performed only during the emission of the laser, the non-emission of the laser, or the irradiation of the laser with a fixed timing.

(Third Embodiment) In the third embodiment, a schematic configuration diagram of a scanning optical microscope to which the light quantity measuring device described in the first embodiment is applied is shown in FIG. Since it is the same, the same figure shall be used.

FIG. 5 shows the photometric circuit 41 (40) and the main controller 5
0 and a schematic configuration diagram of the light irradiation control unit 51 are shown, and the same portions as those in FIG. 2 are denoted by the same reference numerals.

In this case, the photometric circuit 41 (40) further has a time limit setting section 14. The time limit setting unit 14 receives the time limit from the system control unit 11,
The number of limited clocks is calculated accordingly, and when the clock counting unit 4 reaches the number of limited clocks, it is considered that the pulse counting by the pulse counting unit 3 has finished.

In the third embodiment as described above, the flowchart shown in FIG. 6 is executed. In this case, the system control unit 11 sets the set pulse number in the pulse number setting unit 8 (step 601), and then the system control unit 11 inputs the time limit of the time limit setting unit 14 (step 602).

Then, in steps 603 to 606,
Steps 302, 303, 306 shown in FIG.
An operation similar to that of 307 is executed, and it is determined whether the number of clocks counted by the clock counting unit 4 has reached the limit clock number corresponding to the time limit set in the time limit setting unit 14 (step 607). If the number is not reached,
Next, it is judged whether the number of pulses counted by the pulse counting section 3 has reached the set pulse number (step 608), and if it has not reached the set pulse number, the operations of steps 605 to 608 are repeated.

After that, when the number of clocks counted by the clock counting unit 4 reaches the limit number of clocks or the number of counting pulses of the pulse counting unit 3 reaches the set number of pulses, the clock count value at this time is set to the address. The data is stored in the memory 7 according to the designated address input from the control unit 6 (step 609).

Subsequent steps 610 to 614 are the same as steps 311 to 315 shown in FIG. 3 described above.

With such a configuration, it is possible to prevent the fluorescence amount of the observation sample 30 from being so small that it takes a long time to perform pulse counting until the set pulse number is reached.
It is possible to prevent the fading from progressing by irradiating a single place with the laser for a long time.

(Modification of Third Embodiment) The above-mentioned third embodiment
In the embodiment, the displacement of the galvanometer mirrors 25 and 26 occurs when the pulse count value of the pulse counting unit 3 reaches the set pulse number or when the clock count value reaches the limit clock number. The displacement (scanning speed) is always constant, the time limit of the time limit setting unit 14 is set to the scanning speed per pixel, and when the pulse count value reaches the set pulse number within the time limit, the light emission control unit 13
You may make it prevent light irradiation. By doing so, the operation of the galvanometer mirror can be made to be the same as the conventional one, so that the conventional scanning technique of the galvanometer mirror can be used.

(Fourth Embodiment) In the fourth embodiment, a schematic configuration diagram of a scanning optical microscope to which the light quantity measuring device described in the first embodiment is applied is shown in FIG. Since it is the same, the same figure shall be used.

FIG. 7 shows the photometric circuit 41 (40) and the main controller 5
0 and a schematic configuration diagram of the light irradiation control unit 51 are shown, and the same portions as those in FIG. 2 are denoted by the same reference numerals.

In this case, the photometric circuit 41 (40) further includes a charge storage unit 15, a charge amount comparison unit 16, and a stored charge threshold value setting unit 17. The charge storage unit 15 stores the current pulse from the photodetector 1 (1 ') as a charge.
The accumulated electric charge threshold value setting unit 17 receives the setting of the accumulated electric charge threshold value from the system control unit 11. Further, the charge amount comparison unit 16 compares the charge amount of the charge accumulation unit 15 with the charge amount of the accumulated charge threshold value setting unit 17, and the charge accumulation unit 15 is compared.
When the charge amount of the charge reaches the charge amount of the accumulated charge threshold setting unit 17, a signal indicating that the charge accumulation reaches the threshold value is output to the clock counting unit 4, and at the same time, the charge accumulation unit 1
A signal for discharging the charge of No. 5 is output to the charge storage unit 15.

In the fourth embodiment as described above, the flowchart shown in FIG. 8 is executed. In this case, first
The threshold value of the accumulated charge of the accumulated charge threshold value setting unit 17 is input from the system control unit 11 (step 801), then the clock count value of the clock counting unit 4 is initialized (step 802), and the charge is continued. The charges of the storage unit 15 are also discharged and initialized (step 803). The electric current pulse from the photodetector 1 (1 ') is used as electric charge, and the electric charge accumulation unit 15
(Step 804). Further, the clock counting unit 4 counts the number of clocks (step 805).

It is judged whether or not the amount of charges accumulated in the charge accumulating portion 15 has reached the threshold value (step 806). If not, the operations of steps 804 to 806 are repeated.

After that, when the amount of charge stored in the charge storage unit 15 reaches a threshold value, the clock count value used for storing the charge amount up to this point is output to the memory 7 as digital data, and the address control unit The data is stored in the memory 7 in accordance with the designated address input from 6 (step 80
7).

The subsequent steps 808 to 812 are
This is similar to steps 311 to 315 shown in FIG. 3 described above.

With such a configuration, for example, in the photon counting method, current pulses exceeding the threshold value of the broken line are counted as shown in FIG. 9, but as shown in FIG. When the light intensity per unit time increases as shown in Fig. 6 (b) compared to the case where the light intensity is low, the intervals of the current pulses are narrowed and they overlap each other, so that the number of photons may not be accurately counted. By measuring the amount of light with the amount of electric charges of 1, the number of photons can be accurately counted even when the amount of light per unit time is large, and the amount of light can be accurately measured.

(Fifth Embodiment) In this fifth embodiment, a schematic configuration diagram of a scanning optical microscope to which the light quantity measuring device described in the first embodiment is applied is shown in FIG. Therefore, the same drawing is used, and the schematic configuration diagram of the photometric circuit 41 (40), the main control unit 50, and the light irradiation control unit 51 is the same as that in FIG. 5, and thus the same drawing is used.

In this case, the time limit setting unit 14 receives the time limit from the system control unit 11, calculates the number of time limit clocks corresponding to the time limit, and if the clock counter 4 counts the number of time limit clocks, the system control is performed. An instruction to increase the irradiation light amount is given to the light emission control unit 13 through the unit 11,
The system controller 11 causes the memory 7 to store the ratio of the increased irradiation light amount. Then, the clock number of the pixel that has reached the limit clock number is acquired again. Also,
In the data processing unit 9, (reference photon number) / (sampling time) /
The brightness information is calculated by obtaining (ratio of change of irradiation light amount).

In the fifth embodiment as described above, FIG.
The flowchart shown in is executed. In this case, in steps 1001 to 1007, operations similar to those in steps 601 to 607 shown in FIG. 6 described above are executed, and when the clock counting unit 4 counts the limited clock number, the light emission control unit 13 is irradiated with the light amount. Is given (step 1008), and the system controller 11 causes the memory 7 to store the ratio of the increased irradiation light amount (step 1008).
9). Then, returning to step 1003, the same operation as described above is repeated.

After that, when the number of counted pulses of the pulse counter 3 reaches the set number of pulses (step 1010), the clock count value at this time is stored in the memory 7 according to the designated address inputted from the address controller 6 ( Step 10
11).

Then, the light emission control unit 13 is instructed to irradiate the next measurement position on the observation sample 30 with light (step 1012), and the address control unit 6 is further subjected to a memory corresponding to the next measurement position. 7 is designated (step 1013), and then the digital data of the measurement result stored in the memory 7 is read to the data processing unit 9 of the main control unit 50 and used as the luminance information (reference photon number).
/ (Sampling time) / (Ratio of change in irradiation light amount) is calculated (step 1014), and the calculated luminance information is displayed on the display unit 10 (step 1015). Then, it is determined whether the image acquisition is to be ended (1016), and when it is not ended, the above-described operations of steps 302 to 315 are repeated.

With this structure, it is possible to prevent the fluorescence amount of the observation sample 30 from being so small that it takes a long time to perform the pulse counting until the set number of pulses is reached.
The measurement time can be shortened by increasing the irradiation light amount and the fluorescent light amount. In this case, when the irradiation light amount is increased, the fluorescence light amount also changes, but the change can be canceled by the data processing.

(Sixth Embodiment) In the third embodiment, a schematic configuration diagram of a scanning optical microscope to which the light quantity measuring device described in the first embodiment is applied is shown in FIG. Since it is the same, the same figure shall be used.

FIG. 11 shows a schematic configuration of the photometric circuit 41 (40), the main control section 50 and the light irradiation control section 51. The same parts as those in FIG. There is.

In this case, the photometric circuit 41 (40) further has an excess light amount detection section 18. The excess light amount detection unit 18 receives the set time from the system control unit 11,
If the number of clocks of one pixel counted by the clock counter 4 is smaller than the number of clocks set by the excess light amount detector 18, the set number of clocks is calculated according to the time, and the light emission controller 13 is passed through the system controller 11. The system controller 11 causes the system controller 11 to store the ratio of the increase in the irradiation light amount in the memory. Then, the number of clocks of the pixel having the excessive light amount is acquired again. Further, the data processing unit calculates the luminance information by obtaining (reference photon number) / (sampling time) / (ratio of change of irradiation light amount).

In the sixth embodiment as described above, FIG.
The flowchart shown in is executed. In this case, in steps 1201 to 1206, the same operation as steps 1001 to 1006 shown in FIG. 10 described above is executed, and the number of counting pulses of the pulse counting unit 3 must reach the set number of pulses.
(Step 1207) and steps 1205-1207 are repeated. When the number of counting pulses of the pulse counting unit 3 reaches the set number of pulses, the clock counting unit 4 at this time
It is determined whether or not the clock count value counted by is smaller (shorter) than the number of clocks (set time) set by the excess light amount detection unit 18 (step 1208). If the number of clocks is shorter (shorter) than the set number of clocks (set time), the light emission controller 13
To give an instruction to reduce the irradiation light amount (step 120
9) Then, the system controller 11 causes the memory 7 to store the ratio of the reduced irradiation light amount (step 1210). Then, returning to step 1203, the same operation as described above is repeated.

On the other hand, if it is determined in step 1208 that the number of clocks is longer (longer) than the set number of clocks (set time),
Step 1211 and subsequent steps are performed, but in this case, step 1
211 to 1216 are step 1 shown in FIG. 10 described above.
The same as 011 to 1016.

With such a configuration, as shown in FIG. 9B, the amount of light per unit time becomes too large, and at the same time, the probability that photons will be incident on the photodetector 1 will increase, and the photon counting will be accurate. When there is a possibility that the photodetector 1 (1 ') cannot be performed, the amount of irradiation light can be reduced to reduce the amount of fluorescent light, and the state of the photodetector 1 (1') capable of accurately counting photons can be maintained.
Moreover, since it is possible to prevent the irradiation light from being excessively irradiated by reducing the irradiation light amount, it is possible to prevent fading of the observation sample 30. Furthermore, when the irradiation light amount is reduced, the fluorescence light amount also changes, but the change can be canceled by data processing.

In the first to sixth embodiments described above, the laser light source 21 includes the galvanometer mirrors 25 and 2.
Although the observation sample 30 is scanned by 6, the galvanometer mirrors 25 and 26 may be stopped to measure one point of the observation sample 30.

In addition, the present invention is not limited to the above-mentioned embodiment, and can be variously modified at the stage of carrying out the invention without changing the gist thereof.

Furthermore, the above-described embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiment, the problem described in the section of the problem to be solved by the invention can be solved, and it is described in the section of the effect of the invention. In the case where the effect described above is obtained, a configuration in which this constituent element is deleted can be extracted as an invention.

The above-described embodiment also includes the following inventions.

(1) In the light quantity measuring device according to any one of claims 1 to 3, light irradiation control for controlling that the photon counting means reaches a set number of photons and irradiation of the illumination light to the observation sample. The feature is that the parts work together.

By doing so, the measurement of each part of the observation sample and the control of the irradiation light are interlocked, and when the detected light amount reaches a predetermined value at a certain fixed position, the irradiation position of the light is moved to the next measurement position. You can

(2) In the light quantity measuring device according to any one of claims 1 to 3, the maximum sampling time is set, and when the sampling time reaches the maximum time set by the time limit setting section, the photon count value is reached. Is characterized by having a time limit setting unit that performs the same processing as when the set number of photons is reached even if the set number of photons is not reached.

By doing so, it is possible to prevent the number of photons from being small and the illumination light from being applied for too long.

(3) In the light quantity measuring device according to any one of claims 1 to 3, the shortest sampling time and / or the longest sampling time is set, and the sampling time is shorter than the shortest time (longer than the longest time). In this case, the light irradiation control unit has a limit time setting unit that reduces (increases) the irradiation light amount.

By doing so, when the number of photons is too large (too small), the amount of illumination light can be decreased (increased).

(4) In the light quantity measuring device according to any one of claims 1 to 3, when the irradiation light quantity is changed, (set photon number) / (sampling time) / (ratio of change of irradiation light quantity) is calculated. Then, it is characterized by having a data processing unit for calculating the luminance information.

By doing so, it is possible to prevent the brightness information from becoming discontinuous with the brightness information acquired up to that time when the light quantity of the illumination light is changed.

[0080]

As described above, according to the present invention, it is possible to obtain a good image in which the S / N is almost constant in a place where the light amount is large and a place where the light amount is small, and a quick image acquisition speed is obtained. A light quantity measuring device that can be secured can be provided.

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic configuration centering on an optical system of a scanning optical microscope to which a light quantity measuring device according to a first embodiment of the present invention is applied.

FIG. 2 is a diagram showing a schematic configuration of a photometric circuit, a main control unit, and a light irradiation control unit used in the first embodiment.

FIG. 3 is a flowchart for explaining the operation of the first embodiment.

FIG. 4 is a flowchart for explaining the operation of the second embodiment of the present invention.

FIG. 5 is a diagram showing a schematic configuration of a photometric circuit, a main control unit, and a light irradiation control unit used in a third embodiment of the present invention.

FIG. 6 is a flowchart for explaining the operation of the third embodiment.

FIG. 7 is a diagram showing a schematic configuration of a photometric circuit, a main control unit, and a light irradiation control unit used in a fourth embodiment of the present invention.

FIG. 8 is a flowchart for explaining the operation of the fourth embodiment.

FIG. 9 is a diagram for explaining the photon counting method according to the fourth embodiment.

FIG. 10 is a flowchart for explaining the operation of the fifth embodiment of the present invention.

FIG. 11 is a diagram showing a schematic configuration of a photometric circuit, a main control unit, and a light irradiation control unit used in a sixth embodiment of the present invention.

FIG. 12 is a flowchart for explaining the operation of the sixth embodiment.

[Explanation of symbols]

1, 1 '... Photodetector 2. Current-voltage converter 3 ... Pulse counter 4 ... Clock counter 5 ... Clock generator 6 ... Address control unit 7 ... Memory 8 ... Pulse number setting section 9 ... Data processing unit 10 ... Display 11 ... System control unit 12 ... Galvo mirror control unit 13 ... Light emission control unit 14 ... Restriction time setting section 15 ... Charge storage unit 16 ... Charge amount comparison unit 17 ... Accumulated charge threshold setting section 18 ... Excessive light amount detection unit 21 ... Laser light source 22 ... Beam expander 23 ... Wavelength filter 24 ... Optical path splitting means 25.26 ... Galvanometer mirror 27 ... Pupil projection lens 28 ... Prism 29 ... Objective lens 30 ... Observation sample 31 ... Lens 32 ... Mirror 33 ... Lens 34 ... Optical detector 35 ... Frame memory 36 ... Condensing lens 37 ... Dichroic mirror 38 ... Absorption filter 39 ... Pinhole plate 40, 41 ... Photometric circuit 42 ... Mirror 43 ... Dichroic filter 44 ... Absorption filter 45 ... Pinhole plate 50 ... Main control unit 51 ... Light irradiation control unit

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2G043 AA03 AA06 EA01 FA02 GA01                       GA02 GB01 GB19 HA01 HA02                       HA09 HA15 JA02 KA09 LA01                       LA02 NA01 NA05 NA06                 2G065 AA04 AB09 AB11 BA09 BA18                       BB22 BB26 BC01 BC14 BC15                       BC17 BC19 BC22 BD01 BD03                 2H052 AA07 AA09 AC04 AC15 AC27                       AC34 AF02

Claims (3)

[Claims] 1. A light detection means for detecting, as photons, light obtained by irradiating an observation sample with illumination light from a light source or light emitted by the observation sample, and a light amount setting in which a light amount corresponding to a predetermined number of photons is set in advance. Means, a time measuring means for measuring the time until the light quantity corresponding to the number of photons detected by the light detecting means reaches the light quantity set in the light quantity setting means, and a predetermined light quantity set in the light quantity setting means And a brightness information calculating means for calculating brightness information of the observation sample according to the time measured by the time measuring means. 2. Light detection means for detecting, as photons, light obtained by irradiating an observation sample with illumination light from a light source or light emitted by the observation sample, and photons for counting the number of photons detected by the light detection means. Counting means, photon number setting means for setting a predetermined number of photons in advance, and time measurement for measuring the sampling time until the photon count value of the photon counting means reaches the set number of photons set in the photon number setting means. A light quantity measuring device comprising: a means and a brightness information calculation means for calculating brightness information of the observation sample based on the number of photons set by the photon number setting means and the sampling time measured by the time measuring means. 3. Light detection means for detecting light obtained by irradiating an observation sample with illumination light from a light source or light emitted by the observation sample, and an output corresponding to the number of photons detected by the light detection means. Integrating means for integrating a threshold value setting means for setting a predetermined threshold value in advance, and a sampling time until the integrated value of the integrating means reaches the threshold value set for the threshold value setting means. A time measuring means for measuring, and a brightness information calculating means for calculating the brightness information of the observation sample based on the threshold value of the threshold value setting means and the sampling time measured by the time measuring means. Light intensity measuring device.