JP2005234500A - Light receiving device of scanning type laser microscope and scanning type laser microscope using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve such a problem that it is difficult to perform the correction of black level of an image at every one frame, particularly one line scanning and it is made necessary to survey the position corresponding to the black level through image processing upon the high speed scanning because a mechanical operation for light shielding of a photoelectric conversion circuit 1 is necessary in order to fetch the data upon the light shielding in the conventional black level correction system and, moreover, the conventional black level correction system can not be applied to a sample having uniform brightness and is susceptible to the influence of fetching errors of images. <P>SOLUTION: The scanning type image input apparatus has a laser beam source which performs optically two-dimensional scanning of the sample and can be controlled so as not to apply laser on the position outside visual field such as retrace line interval, receives the obtained beam through the photoelectric conversion circuit and converts the same into a brightness signal and obtains the information of a sample image by subjecting the brightness signal to the image processing. Therein, by using the retrace line where the beam from the sample is not made incident into the photoelectric conversion circuit, a correction amount of the black level of the image is detected from the signal included in the output of the photoelectric conversion circuit in the state of optical amount 0 and the brightness signal is corrected based on the detected correction amount. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to scanning image input for forming a sample image by scanning a sample two-dimensionally with light, and more particularly to a confocal scanning optical microscope.

  An optical microscope has a structure in which a specimen on a preparation placed on a stage is observed by magnifying it with an objective lens. In general, illumination of a specimen uses light from a light source such as a lamp to observe the specimen using a condenser lens. A structure in which illumination is performed so that the entire area is uniform is employed. However, when such a structure is adopted as the illumination system, there are problems such as flare, and there is a problem that it is very difficult to observe when observing a low-contrast sample.

  In order to improve these problems, a confocal scanning optical microscope, which is a point light projection type (spot light projection type) optical microscope, has been proposed. This confocal scanning optical microscope irradiates an observation specimen with a point light source such as a laser light source in a point shape via an objective lens, thereby irradiating the observation specimen with transmitted light, reflected light or spot light. The generated fluorescence is imaged in the form of a dot again through the objective lens and the optical system, and this is detected by a detector to obtain the density information of the image.

  Since only the intensity information of the point irradiated with the point light source can be obtained by this alone, an xy scanning method in which the specimen is moved in the x-axis and y-axis directions and mechanically moved in the two-dimensional plane, or an optical path A scanning optical system that scans and scans the specimen and the irradiation point position relatively two-dimensionally, and each point corresponding to the xy scanning position is synchronized with the xy scanning by a display device such as a CRT display. By displaying a bright spot corresponding to the signal of the density information at the position, the image can be observed.

  In recent years, retrace time of two-dimensional scanning using laser light sources and laser diodes using acousto-optic elements, etc. so that point light sources are used only in places necessary for observing biological specimens and measurement is not wasted. Alternatively, there is a control in which an excitation light source is not applied to a region other than a specified region.

  The above is the principle configuration of the confocal scanning optical microscope. Specifically, A / D is obtained by converting transmitted light, reflected light, or fluorescence from a specimen scanned with laser light into an electrical signal by a photoelectric converter such as a photomultiplier tube or a photodiode as a detector. After being quantized by the conversion circuit, it is stored in the memory. As described above, the confocal scanning optical microscope includes a photoelectric signal processing circuit that converts sample image information into an electric signal by a photoelectric converter, converts the sample image information into a digital signal, and stores the digital signal in a memory.

  In particular, photoelectric conversion circuits convert minute light intensities into electrical signals, so there is an integration method that amplifies the voltage to be measured by accumulating charges generated in photomultiplier tubes and photodiodes in capacitive elements such as capacitors. It is taken.

  FIG. 3 shows a circuit configuration until the analog signal photoelectrically converted in the confocal scanning optical microscope is quantized. Light incident on the photoelectric conversion circuit 1 is converted into an electric signal by the photoelectric conversion circuit 1, and the electric signal is quantized by the A / D conversion circuit 2 and then sent to the control unit as a digital value. Further, this digital value is imaged after being subtracted by the digital subtractor 4.

  For example, when scanning the specimen shown in FIG. 4, if the optical system of the confocal scanning optical microscope is focused on the highest surface of the specimen shown in FIG. A signal as shown in (c) is generated. That is, the signal level is high because the amount of light incident on the photoelectric conversion circuit 1 increases on the in-focus surface, and the signal level becomes 0 in the scanning region before and after the in-focus surface because there is no light amount incident on the photoelectric conversion circuit 1. Become. The portion where the signal level becomes low becomes the black level of the final image. The black level in the image corresponds to the output of the photoelectric conversion circuit 1 when no light is incident on the photoelectric conversion circuit, and is an offset voltage in the circuit.

  In general, the black level of an image varies depending on the temperature dependence of the output of a photoelectric conversion element or an operational amplifier used in a photoelectric conversion circuit. Therefore, paying attention to the scanning image input device and scanning probe microscope output of the digital subtraction circuit 4, the black level of the image from the first line to the nth line is scanned during scanning of a plurality of lines as shown in FIG. It has fluctuated. Therefore, the black level is corrected after the data acquisition for the nth line is completed. In FIG. 5, the vertical axis represents the digital subtraction circuit output converted into an analog value.

The above black level correction is realized by capturing the data with the incident light of the photoelectric conversion circuit 1 blocked by a method such as closing the shutter, and subtracting the data at the time of shading from the current input data in the digital subtractor 4. Is done. Further, since the black level correction method as described above requires a mechanical operation to shield the photoelectric conversion circuit 1 in order to capture data at the time of light shielding, an image is captured every frame, particularly every line scanning, during high-speed scanning. It is difficult to perform black level correction. For this reason, the amount of correction of the black level of the image is detected from the signal included in the output of the photoelectric conversion circuit using the portion where the light from the sample does not enter the photoelectric conversion circuit during the scanning time, and the detected correction A configuration in which the luminance signal is corrected based on the amount is also considered.
JP-A-9-243929 JP-A-9-297269

  However, since the black level correction method as described above requires a mechanical operation to shield the photoelectric conversion circuit 1 in order to capture the data at the time of light shielding, the image is captured every frame, particularly every line scanning, during high-speed scanning. It is difficult to correct the black level, or it is necessary to search for a position corresponding to the black level by image processing. In addition, it cannot be applied to a sample with uniform brightness, and image capture errors Also susceptible to.

  The present invention has been made in view of the above circumstances, and can block the incident light to the photoelectric conversion circuit at high speed during sample scanning, thereby correcting the black level of the image, and confocal with high scanning speed. An object of the present invention is to provide a scanning microscope that can realize black level correction of a real-time image even in a scanning optical microscope and obtain an image with a stable black level.

  In order to achieve the above object, the present invention takes the following measures. The present invention has a laser light source that can optically two-dimensionally scan a sample and can be controlled so as not to irradiate the laser at a position outside the visual field such as a blanking interval. In a scanning type image input device that receives light by a photoelectric conversion circuit and converts it into a luminance signal, and performs image processing on the luminance signal to acquire information on the sample image, light from the sample enters the photoelectric conversion circuit during the scanning time. No (time when image data is not acquired) Using the blanking interval, the black level correction amount of the image is detected from the signal included in the output of the photoelectric conversion circuit in the state of light amount 0, and this detected correction The luminance signal is corrected based on the amount.

  Figure 1 shows the basic configuration of the laser microscope. The laser light source 11 that irradiates the specimen 18 with excitation light has a feature capable of ON / OFF control of the light source in units of at least several μs. The excitation light emitted from the light source is converted into an objective lens by the two-dimensional scanning device 14. The sample is irradiated with laser through 17. At this time, the laser beam performs a raster scan as shown in FIG. 6 by the two-dimensional scanning device 14. In this raster scan, after moving in one direction on the plane, the movement to the original position is performed. This time becomes a retrace time, and irradiating the sample 18 with a laser gives an unnecessary change, so the laser irradiation is turned off.

  Fluorescence (or reflected light) generated by the excitation light applied to the specimen 18 passes through the objective lens 17 and is introduced into the detection optical path. In the detection optical path, after passing through a wavelength selection filter or pinhole, only the necessary wavelength is incident on the light receiving element 41.

  The light receiving element 41 performs photoelectric conversion according to the intensity of incident light, and generates a current (charge). This amount of current (total amount of charges) has a correlation with the intensity of light. In order to construct the sample surface information as two-dimensional information, the current amount is measured at regular intervals, and imaged in association with the laser irradiation position.

  A circuit for measuring the amount of current (FIG. 9) includes a light receiving element 41 that receives measurement light, a capacitive element 42 that receives current from the light receiving element 41, a discharge circuit 43 that discharges charges accumulated in the capacitive element 42, a discharge Consists of an analog switch 45 that controls the circuit 43, an A / D converter circuit 46 that measures the charge accumulated in the capacitive element as a potential difference, and a circuit that sends digital data from the A / D converter 46 to the image display device as measurement information Has been.

  Since the circuit for measuring the amount of current has a very small current to be measured of several hundred nA or less, an analog integrating circuit is used for the purpose of removing noise and the purpose of amplification. In the analog integrating circuit, the current from the light receiving element 41 accumulates electric charge in a capacitive element and is measured in the form of a potential difference between both ends of the capacitive element. Since this potential difference is due to the accumulation of electric charges, the potential difference increases as it accumulates for a long time, and the noise contained in the current is also averaged. After the potential difference is measured by an A / D converter or the like, the charge accumulated for measurement at the next position is connected to the discharge circuit 43 by the analog switch 45 and discharged.

  Here, since the amount of charge cannot be zero unless a sufficient time is taken for discharging the charge, it is necessary to set a sufficient discharge time. Further, it is possible to have a circuit configuration in which charges are positively discharged by devising the discharge circuit 44 and discharged to a constant voltage in a short time, but the circuit elements are complicated. Furthermore, generally, the constituent elements of the electric circuit have temperature characteristics, and the analog integration circuit has temperature characteristics inside the discharge circuit, so the discharge amount has temperature characteristics. For this reason, when the ambient temperature changes, the discharge amount for each pixel changes, and as a result, the black level fluctuates on the display screen, and information that does not have stripes is mixed into the sample image.

  In order to prevent such a phenomenon, it is very difficult to perform temperature compensation in a circuit, so measure a certain point of data as a reference value and subtract the reference value from the digital data after A / D conversion. Correction processing is performed.

  The reference value data for correcting the black level needs to be measured in a state where the amount of light is 0, and it is necessary to frequently measure and correct in order to cope with fluctuations in a short time. Therefore, the blanking time in the horizontal direction or the vertical direction generated when the sample is scanned two-dimensionally is used. During this time, measurement of data necessary for image display is not performed, and if the device uses a laser light source capable of high-speed on / off control, the laser light is turned off only during this time and the amount of light from the sample is set to 0. can do. The black level data necessary for this time is acquired. The data acquisition method is the same as the measurement of luminance information from a normal light receiving element.

  In the case of claim 2, in order to measure faint fluorescence, if the integration time per pixel is very long, if the black level measurement is measured under the same sampling time conditions, it may not be completed within the retrace time. Occur. The black level measurement itself is theoretically accumulated no matter how long it is accumulated, and the accumulated charge from the light receiving element is 0. Therefore, no problem occurs even if the measurement cycle is shortened. For this reason, the sampling speed is changed only for the measurement of the black level, and the operation is performed so that the data is reliably measured within the blanking time and the image capturing time is not affected.

  In the case of claim 3, the amount of charge accumulated in the black level measurement is theoretically 0 in the previous paragraph, but in an actual circuit, there is an offset voltage generated in the circuit element, and a leakage current associated therewith. The black level reference changes as shown in FIG. For this reason, the measurement standard is measured at the start of accumulation immediately after the end of discharge, which does not affect the accumulation.

  In the case of claim 5, since the coefficient of temperature exists in the change of the black level, the correction amount is calculated by measuring the temperature of a thermistor or the like.

  In the case of claim 6, when there is an actual image data sampling speed and separation due to the fluctuation of the black level reference, the black level offset cannot be completely removed, and the black level brightness of the image is changed every time the image sampling speed is changed. Happens to change. Therefore, the correction amount is adjusted by measuring the sampling rate and the change amount of the black level in advance and obtaining the correlation.

  According to the configuration of claim 1, according to this method, the black level can be obtained by controlling the laser light source without using a mechanical operation such as a mechanical operation. In addition, since correction values can be acquired in a short time, correction can be performed in units of lines, and horizontal stripes due to black level fluctuations do not occur in the sample image displayed on the screen.

  According to the second, third, third and fourth aspects of the present invention, data can be acquired under conditions other than normal image measurement, so that various correction data processing methods can be used, such as averaging.

  According to the configuration of claim 5, it is possible to use an appropriate black level correction value even for a temperature change.

According to the configuration of claim 6, correction can be performed by estimating the correction value without a temperature sensor or the like.

  Example 1 will be described with reference to the drawings. Laser light source 11 and acousto-optic device that performs high-speed laser ON / OFF control, a scanning device consisting of a two-axis galvanometer mirror that scans the sample surface in two dimensions, and the amount of fluorescence or reflected light from the sample surface In a laser scanning microscope comprising a light receiving circuit and a control unit for imaging a light intensity signal from the light receiving circuit and controlling ON / OFF of the laser and two-dimensional scanning, the light receiving circuit is a photomultiplier tube or a photodiode Light receiving element 41, capacitor 42 that stores photoelectrically converted current, AD converter 46 that converts the potential difference between both ends of the capacitor, discharge circuit 43 that discharges the accumulated charge, and analog switch that switches the discharge circuit It consists of 45 circuits.

  The control unit 54 determines whether the laser light emitted from the laser light source 11 is irradiated at a retrace time, and controls the acoustooptic device to turn off the laser light when it is the retrace time. The laser beam that has passed through the acousto-optic device is irradiated onto a biaxial galvanometer mirror, and the sample surface is scanned two-dimensionally. The laser light applied to the sample surface generates fluorescence or reflected light, and again passes through the galvanometer mirror and is guided to the light receiving circuit. The control unit 54 receives the light amount data from the light receiving circuit when the laser comes to the point to be imaged while managing the position of the two-dimensional scanning. The received data is collected into line or surface data and displayed on the display.

  The light receiving circuit photoelectrically converts light from the sample by a light receiving element 41 such as a photomultiplier tube or a photodiode, and the converted current is accumulated (integrated) by a capacitor to be a potential difference between both ends of the capacitor. The potential difference is detected by the AD converter 46, converted into digital data, and sent to the control unit for imaging. In addition, after the measurement of the A / D converter 46 is completed, the charge accumulated in the capacitor 42 is connected to the discharge circuit 43 by the analog switch 45 for measurement at the next point, and the accumulated charge is discharged.

  During the measurement of the specimen surface as described above, the black level is measured during the blanking time in the horizontal or vertical direction during two-dimensional scanning. Since the laser beam is blocked by the acoustooptic device when the retrace time is reached, the light from the sample surface becomes zero. The black level is measured at this timing.

The black level data taken in the same manner as the other light quantity data is taken into the control unit, and the light intensity data from the subsequent specimen is subtracted for black level correction. The black level correction value is constant until the next black level measurement is performed.
According to this method, the black level can be acquired by controlling the laser light source without using a mechanical operation such as a mechanical operation. In addition, since correction values can be acquired in a short time, correction can be performed in units of lines, and horizontal stripes due to black level fluctuations do not occur in the sample image displayed on the screen.

As a modification, the laser light source is an ordinary CW laser connected with an acousto-optic device, which achieves high-speed on / off control. In addition to the acousto-optic device, a device with microsecond order response such as EOM or liquid crystal shutter You may do it. Alternatively, a laser light source using a laser diode capable of high-speed on / off control alone may be used. The control unit 54 is composed of a dedicated circuit centered on a personal computer used for image display, but the control up to correction control may be housed in a dedicated digital circuit.
Although the discharge circuit is the simplest CR discharge circuit, it may be a charge circuit that sucks out (adds) charges so as to have a constant potential.

  Example 2 will be described. In the configuration of the first embodiment, a method for switching the conditions for obtaining the black level correction value is provided. In addition to the configuration of the first embodiment, the control unit includes the A / D converter 46 and the analog switch 45 in the light receiving circuit. The operating frequency can be changed arbitrarily during measurement.

  When the normal pixel-by-pixel measurement is finished and the retrace time is reached, the sampling speed of the light receiving circuit is changed to a sampling speed for black level measurement. The black level is then measured at the changed sampling rate. For this reason, the operation of measuring the potential after a predetermined time from the completion of discharge is performed regardless of the sampling rate of the image data. As a result, the black level can be measured under the same conditions even if the sampling rate is changed and an image is acquired.

  In the method of the first embodiment, black level data is acquired at the same sampling interval as that for normal pixel-by-pixel data. However, when this sampling interval becomes longer, the black level measurement may not end within the baseline time. It happens. In such a case, the measurement can be completed within the baseline time by changing the speed only during the black level measurement.

  Furthermore, since data after a certain time from the completion of the discharge can be taken without fail, the charge due to the charge flowing into the capacitor 52 from the outside can be specified. In particular, since there is a transient response of the potential at the moment of changing from discharge to accumulation, it is possible to obtain data by avoiding such a portion.

  As a modification, since the sampling speed can be changed, the black level measurement may be sampled a plurality of times within one retrace time, and an arithmetic process such as averaging may be performed to obtain a correction value.

  Example 3 will be described. In addition to the configuration of the second embodiment, the thermistor temperature measuring means is provided around the light receiving circuit, and the control unit retains the black level temperature characteristic, and temperature change occurs by obtaining temperature information from the temperature measuring means. This is a light receiving circuit in which an appropriate correction value can be obtained even in the case of an error.

  The current that does not partition the discharge, which is the cause of black level fluctuations, or the charge that leaks from the outside of the field is temperature dependent. For this reason, as shown in FIG. 8, the black level (offset amount) after a predetermined time has changed. Here, in the method of the second embodiment, the data after a certain time from the completion of the discharge is set to the black level. Therefore, when the actual image data acquisition time and the capture timing are different, there is a difference in the black level amount. Therefore, in such a case, it is necessary to determine the correction value in consideration of the temperature change amount.

  The thermistor is arranged around the light receiving circuit, particularly the capacitor 42 and the discharge circuit 43 related to the temperature coefficient. The thermistor that detects the temperature of the light receiving circuit is taken into the control unit via the conversion circuit. The control unit has in advance the relationship between the sampling speed and the black level due to temperature changes as table data or a function, and the optimum correction value for the sampling speed is obtained from the temperature information from the thermistor and the current black level measurement value. Obtained and used for image processing.

  As a modification, the temperature measuring means may use a temperature sensitive resistor in addition to the thermistor.

  Example 4 will be described. In addition to the configuration of the second embodiment, the control unit holds the temperature characteristics of the black level for each arbitrary time, and the temperature information is estimated by measuring several points of the black level by changing the time. Even if it occurs, the light receiving circuit can obtain an appropriate correction value.

  As described in the third embodiment, the temperature affects the amount of charge accumulated in the capacitor 42. However, it is easy to estimate the black level after a lapse of a certain time as shown in FIG. 8 by measuring several black levels within a minute time with little temperature change, by performing numerical analysis.

  The control unit measures the black level after elapse of a certain number of points such as 100 ns, 500 ns, 1000 ns, etc. from the completion of the discharge of the charge accumulated in the capacitor 42 during the retrace time. The control unit further obtains a correction function necessary for obtaining the black level after the lapse of the time not measured, such as linear interpolation, least square interpolation, or spline interpolation, from the measured black level data for each time. From this correction function, the black level in the sampling period at the time of image data acquisition is obtained and used for image correction.

  In this method, if the characteristics of the black level fluctuation at the time of temperature change are known, an optimum interpolation function is derived, so that the black level correction value can be obtained with a certain degree of accuracy even without a temperature sensor.

It is a figure which shows the basic composition of a laser microscope. It is a figure which shows the image signal in the case of being focused on the highest surface of a sample. It is a figure which shows the circuit structure until it quantizes the analog signal photoelectrically converted. It is a figure which shows the example of the sample of a confocal scanning optical microscope. It is a figure which shows that the black level of an image is fluctuate | varied between the scanning of several lines. It is a figure which shows the raster scan by a two-dimensional scanner. It is a figure which shows the change of the reference | standard of a black level by long-time accumulation | storage by leak current. It is a figure which shows the change of the black level (offset amount) after progress for a fixed time. It is a figure which shows the circuit which measures an electric current amount.

Explanation of symbols

1: Photoelectric conversion circuit 2: A / D conversion circuit

4: Digital subtraction circuit

11: Laser light source 12: Collimator lens 13: Dichroic mirror 14: Two-dimensional scanning device 15: Lens 16: Dichroic mirror 17: Objective lens 18: Sample 19: Stage

21a: barrier filter 22a: condenser lens 23a: light receiving element 24: condenser lens 25: beam splitter 26: ring slit 27: light receiving element 28: observation optical system 29: transmission illumination optical system 30: incident illumination optical system

41: Light receiving element 42: Capacitive element 43: Discharge circuit

45: Analog switch 46: A / D converter circuit


54: Control unit

Claims (7)

A laser light source unit capable of ON / OFF control,
A scanning device for two-dimensionally scanning laser light onto the sample surface;
A light receiving circuit for measuring the amount of fluorescence or reflected light from the specimen;
In the laser scanning microscope consisting of a control unit that images the light intensity signal from the light receiving circuit and controls the ON / OFF control of the laser and the two-dimensional scanning,
The light receiving circuit includes a light receiving element,
A capacitor for accumulating photoelectrically converted current;
An AD converter that converts the potential difference across the capacitor;
A discharge circuit for discharging the accumulated charge;
A light receiving circuit composed of a circuit comprising a switch for switching a discharge circuit,
A light receiving device characterized by measuring a reference value of luminance 0 by cutting off a laser outside a data acquisition time during scanning of a reference value of display image luminance. 2. The light receiving device according to claim 1, wherein the control unit can set and switch the image data capturing period and the capturing period when measuring the reference value independently. 3. The light receiving device according to claim 2, wherein the control unit measures the reference value immediately after the accumulation of the charge accumulated in the capacitor is started. 3. The light receiving device according to claim 1, wherein the control unit obtains a reference value by performing measurement processing and performing arithmetic processing a plurality of times outside the data acquisition time. 3. A light receiving device comprising a temperature measuring means of a light receiving circuit in addition to the configuration of claim 2, wherein the control unit performs a calculation for correcting a temperature change of the reference value. 4. The light receiving device according to claim 3, wherein the control unit can calculate and obtain a reference value from a plurality of measurements. A laser scanning microscope using the light receiving device according to claim 1.

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