JP2015018070A - Scanning laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a scanning laser microscope capable of consistently acquiring high-precision images.SOLUTION: A scanning laser microscope includes; a scanner; an object lens; an HPD 31 which detects fluorescence and generates a light intensity signal in accordance with a total gain that depends on a plurality of applied voltages; an image generator unit 5 which converts the light intensity signal into luminance information for each pixel corresponding to a scanning position of the scanner to generate an image; a gain input unit 33 for entering a total gain; gain setting units 32A, 32B which set individual gains for applied voltages in the HPD 31; an information storage unit 35 which stores a map comprising a set of individual gains associated with a set of applied voltages for each of the gain setting units 32A, 32B, where each individual gain of the gain setting units 32A, 32B is associated with each total gain with a priority given to a gain setting unit having a higher S/N ratio; and a parameter control unit 37 which reads each individual gain associated with an entered total gain from the map and sets an applied voltage associated with the individual gain in each of the gain setting units 32A, 32B.

Description

  The present invention relates to a scanning laser microscope.

  Conventionally, a scanning laser microscope provided with a photomultiplier tube (PMT) as a detector is known (see, for example, Patent Document 1). The scanning laser microscope described in Patent Document 1 sets the total gain of the PMT based on the voltage applied to the PMT and the circuit gain each time the scan speed is changed, and the same even if the scan speed is changed. A bright image can be obtained.

JP 2005-215357 A

  However, although the total gain of the PMT depends on a single applied voltage, a hybrid detector (HPD) that has recently been used as a detector has a voltage applied to the photocathode and an avalanche diode (AD). The total gain depends on the voltage applied to the two, and there are two solutions for the gain distribution for these applied voltages.

  For this reason, when a detector whose total gain depends on a plurality of applied voltages, such as a hybrid detector, is employed, even if an attempt is made to set an optimum total gain corresponding to the scan speed with reference to Patent Document 1, a plurality of applied It is difficult to properly allocate the gain to the voltage. In addition, even if the total gain is the same, there is a problem in that noise generation differs depending on the gain distribution for a plurality of applied voltages, and the accuracy of the acquired image differs.

  The present invention has been made in view of the above-described circumstances, and provides a scanning laser microscope that can always acquire a highly accurate image when the total gain of a detector depends on a plurality of parameters. With the goal.

In order to solve the above problems, the present invention employs the following means.
The present invention provides a scanning unit that two-dimensionally scans a sample with laser light emitted from a light source, an objective lens that irradiates the sample with laser light scanned by the scanning unit, and a total that depends on a plurality of parameters. According to the gain, a light detection unit that detects return light returning from the sample irradiated with laser light and outputs a light intensity signal corresponding to the luminance of the return light; and a light intensity signal output from the light detection unit. An image generation unit that converts the luminance information for each pixel corresponding to the scanning position of the scanning unit to generate an image of the sample, a gain input unit that inputs the total gain desired by the user, and the light detection unit A plurality of gain setting units for setting individual gains of the respective parameters, and the individual gains and the parameters are associated with the gain setting units, and the gain setting units have a predetermined priority order. A map storage unit for storing a map in which the individual gains of the gain setting units are associated with the total gain, and the gains associated with the total gain input by the gain input unit. Provided is a scanning laser microscope comprising: a parameter control unit that reads the individual gain of a setting unit from the map and sets the parameter corresponding to the read individual gain in each gain setting unit.

  According to the present invention, when laser light is emitted from the light source, it is scanned by the scanning unit and irradiated on the specimen by the objective lens. Then, the return light from the sample is detected by the light detection unit according to the total gain depending on the plurality of parameters and converted into a light intensity signal, and the image generation unit based on the luminance information corresponding to the light intensity signal. An image is generated.

  In this case, when the user inputs the total gain to the gain input unit, the parameter control unit is associated with the total gain according to a predetermined priority order between the gain setting units according to the map stored in the map storage unit. By setting the parameter corresponding to the individual gain of each gain setting unit in each gain setting unit, the light detection unit detects the return light according to the total gain obtained by combining multiple parameters with appropriate individual gains, and the light intensity A signal can be obtained. Therefore, a high-accuracy image can always be acquired by simply inputting a desired total gain using a detector whose total gain depends on a plurality of parameters.

  The present invention provides a scanning unit that two-dimensionally scans a sample with laser light emitted from a light source, an objective lens that irradiates the sample with laser light scanned by the scanning unit, and a total that depends on a plurality of parameters. According to the gain, a light detection unit that detects return light returning from the sample irradiated with laser light and outputs a light intensity signal corresponding to the luminance of the return light; and a light intensity signal output from the light detection unit. An image generation unit that converts the luminance information for each pixel corresponding to the scanning position of the scanning unit to generate an image of the sample, a gain input unit that inputs the total gain desired by the user, and the light detection unit A plurality of gain setting units for setting individual gains for each of the parameters, and the individual gains of the gain setting units according to a predetermined priority order between the gain setting units. A map storage unit that stores the associated map, and the individual gain of each gain setting unit that is associated with the total gain input by the gain input unit is read from the map, and the read individual gain is A scanning laser microscope in which the individual gain and the parameter are associated with each other according to a predetermined function for each gain setting unit. I will provide a.

  According to the present invention, it is not necessary to associate the individual gain and the parameter on the map by associating the individual gain and the parameter on the basis of the predetermined function for each gain setting unit. Therefore, the map can be simplified.

  In the above configuration, the parameter includes a scanning speed input unit for a user to input a scanning speed of the scanning unit, and a scanning speed setting unit for setting the scanning speed input by the scanning speed input unit to the scanning unit, When the scanning speed of the scanning section is reduced according to the ratio of the scanning speed input to the scanning speed input section and the current scanning speed of the scanning section, the control section is input to the gain input section. When the individual gains are read from the map based on a total gain smaller than the total gain, and the scanning speed of the scanning unit is increased, the total gain is larger than the total gain input to the gain input unit. The individual gains may be read from the map.

With this configuration, the scanning unit is scanned at the scanning speed input by the user through the scanning speed input unit and set by the scanning speed setting unit.
In this case, when the scanning speed is slowed down in accordance with the ratio between the input scanning speed and the current scanning speed of the scanning unit, the parameter control unit controls the gain setting unit based on the total gain smaller than the input total gain. Each individual gain is set. Therefore, it is possible to reduce the total gain of the photodetector and prevent the image from being saturated even if the exposure time is increased.

  On the other hand, when the scanning speed increases, the individual gain for each gain setting unit is set by the parameter control unit based on the total gain larger than the input total gain. Therefore, the total gain of the photodetector can be increased, and the brightness of the image can be ensured even when the exposure time is shortened.

  The present invention provides a scanning unit that two-dimensionally scans a sample with laser light emitted from a light source, an objective lens that irradiates the sample with laser light scanned by the scanning unit, and a total that depends on a plurality of parameters. According to the gain, a light detection unit that detects return light returning from the sample irradiated with laser light and outputs a light intensity signal corresponding to the luminance of the return light; and a light intensity signal output from the light detection unit. An image generation unit that converts the luminance information for each pixel corresponding to the scanning position of the scanning unit to generate an image of the sample, a gain input unit that inputs the total gain desired by the user, and the light detection unit A plurality of gain setting units for setting individual gains for each of the parameters, priority information between these gain setting units, settable range information for individual gains that can be set for each gain setting unit, and An information storage unit that stores relational expression information that associates the individual gain and the parameter for each gain setting unit, and the priority order information, the settable range information, and the relational expression information that are stored in the information storage unit Based on the calculation unit, the individual gains of the gain setting units are combined so as to satisfy the total gain input by the gain input unit, and the parameter corresponding to the combined individual gain is calculated, and the calculation unit Provided is a scanning laser microscope comprising a parameter control unit that sets the calculated parameter in each gain setting unit.

  According to the present invention, information on priorities between gain setting units, individual gain settable range information that can be set for each gain setting unit, and relational expression information that associates individual gains and parameters for each gain setting unit in advance. If it is stored in the storage unit, only the total gain is input from the gain input unit, and the parameter control unit gives priority to the desired gain setting unit and satisfies the total gain, and each gain setting unit has an individual gain. Corresponding parameters can be set.

In the above configuration, the predetermined priority may be given priority to the gain setting unit having a high S / N.
By comprising in this way, a light intensity signal with high S / N can be obtained and a more highly accurate image can be acquired.

In the above configuration, the light detection unit may be a hybrid detector, and a voltage applied to the photocathode of the hybrid detector and a voltage applied to the semiconductor element may be used as the parameters.
With this configuration, it is possible to always obtain a highly accurate image by the hybrid detector with a simple operation in which the user simply inputs the total gain.

  According to the present invention, when the total gain of the detector depends on a plurality of parameters, there is an effect that a highly accurate image can always be acquired.

1 is a schematic configuration diagram of a scanning laser microscope according to a first embodiment of the present invention. It is a block diagram of the detection system of FIG. It is a figure which shows the map with which the information storage part of FIG. 2 is provided. It is a figure which shows the relationship between the individual gain and parameter which were matched based on the predetermined function. It is a block diagram of the detection system of the scanning laser microscope which concerns on 2nd Embodiment of this invention.

[First Embodiment]
Hereinafter, a scanning laser microscope according to a first embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, the scanning laser microscope 100 according to the present embodiment includes a stage 1 on which a sample S is placed, a light source 3 that emits laser light, and laser light emitted from the light source 3 on the sample S. , An observation optical system 10 having a scanner (scanning unit) 13 for scanning, a detection optical system 20 and a detection system 30 for detecting fluorescence (return light) generated in the specimen S, and a specimen from which fluorescence is detected by the detection system 30 And an image generation unit 5 that generates an S image.

  The scanning laser microscope 100 also has a control device (scanning speed setting unit) 7 that controls the light source 3, the scanner 13, and the like, and the user gives instructions such as the wavelength of the laser light generated from the light source 3 and the scanning speed of the scanner 13. An input device (scanning speed input unit) 9 for inputting is provided.

  The observation optical system 10 includes a dichroic mirror 11 that reflects the laser light emitted from the light source 3, a scanner (scanning unit) 13 that deflects the laser light reflected by the dichroic mirror 11 and scans it on the sample S, and a scanner. The pupil projection lens 15 that condenses the laser light deflected by 13, the reflection mirror 16 that reflects the laser light condensed by the pupil projection lens 15, and the laser light reflected by the reflection mirror 16 is converted into parallel light. And an objective lens 19 that irradiates the specimen S with laser light converted into parallel light by the imaging lens 17 and collects fluorescence generated in the specimen S.

  As the scanner 13, for example, a galvanometer mirror, a resonant scanner, an AOD (Acousto-Optic Defector), or the like can be used.

  The dichroic mirror 11 reflects the laser light from the light source 3, and allows the fluorescent light that is collected by the objective lens 19 to return in the reverse direction of the laser light to pass through and is branched from the optical path of the laser light. .

  The detection optical system 20 includes a condensing lens 21 that condenses the fluorescence branched from the optical path of the laser light by the dichroic mirror 11, a pinhole 23 that allows a part of the fluorescence condensed by the condensing lens 21 to pass through, And a collimating lens 25 that converts the fluorescence that has passed through the pinhole 23 into parallel light and enters the detection system 30.

  As shown in FIG. 2, the detection system 30 includes a hybrid detector (Hybrid Photo Detector) 31 that detects fluorescence incident from the collimator lens 25 and outputs a light intensity signal, and a user sets a desired total gain of the hybrid detector 31. A gain input unit (GUI) 33 for inputting, an information storage unit (map storage unit) 35 for storing a table (map), and a parameter control unit 37 for controlling parameters of the hybrid detector 31 are provided.

  The hybrid detector 31 includes a photocathode (photodetection unit, not shown) that receives fluorescence and converts it into electrons, and an avalanche diode (AD, photodetection unit) that outputs a light intensity signal corresponding to the electrons converted by the photocathode. , Not shown), a photocathode gain setting unit 32A for setting individual gains of applied voltages (parameters) applied to these photocathodes, and an AD gain for setting individual gains of applied voltages (parameters) to be applied to avalanche diodes. And a setting unit 32B.

  The hybrid detector 31 detects and photoelectrically converts fluorescence according to the total gain depending on the applied voltages (parameters) of the photocathode and the avalanche diode, and generates a light intensity signal having a magnitude corresponding to the amount of fluorescence. To send to. The total gain is the product of the individual gain of the applied voltage applied to the photocathode and the individual gain of the applied voltage applied to the avalanche diode.

  As shown in FIG. 3, the information storage unit 35 includes the table 1 in which each individual gain of the photocathode gain setting unit 32A is associated with the applied voltage, and each individual gain and applied voltage of the AD gain setting unit 32B. And a table 2 associated therewith. In each table 1 and table 2, the applied voltage is an arbitrary value.

  In these tables 1 and 2, the higher the S / N of the photocathode gain setting unit 32A and the AD gain setting unit 32B, for example, the photocathode gain setting unit 32A is given priority over the AD gain setting unit 32B, and the gain Each individual gain between the setting units 32A and 32B is associated with each total gain and stored in the information storage unit 35 as a map.

  In the example of FIG. 3, for example, when the total gain is 300, the individual gain of the photocathode gain setting unit 32A having a high S / N is 300 (table 1), and the individual gain of the AD gain setting unit 32B is 1 (table). 2) is associated. For example, when the total gain is 1500, the individual gain of the photocathode gain setting unit 32A having a high S / N is 1500 (table 1), and the individual gain of the AD gain setting unit 32B is 1 (table 2). It is attached.

  When the total gain is input to the gain input unit 33, the parameter control unit 37 reads the individual gain and AD of the photocathode gain setting unit 32A associated with the total gain from the map stored in the information storage unit 35. The individual gain of the gain setting unit 32B is read out.

  The parameter control unit 37 sets the applied voltage corresponding to the individual gain of the photocathode gain setting unit 32A read from the map in the photocathode gain setting unit 32A, and the individual gain of the AD gain setting unit 32B read from the map. The applied voltage corresponding to is set in the AD gain setting unit 32B. By following the map stored in the information storage unit 35, the photonic surface gain setting unit 32A having a high S / N is prioritized over the AD gain setting unit 32B and the total gain is distributed.

  The image generation unit 5 converts the light intensity signal sent from the hybrid detector 31 into luminance information for each pixel corresponding to the scanning position of the laser beam by the scanner 13 to generate an image of the sample S. Yes. A monitor 6 for displaying the generated image is connected to the image generation unit 5.

  The control device 7 controls ON / OFF of the generation of laser light from the laser 3 and controls the scanning speed and scanning position of the laser light by the scanner 13. In addition, the control device 7 is configured to send scanning position information indicating the scanning position of the laser light to the image generation unit 5.

The operation of the scanning laser microscope 100 configured as described above will be described.
In order to acquire an image of the specimen S with the scanning laser microscope 100 according to the present embodiment, first, the user places the specimen S on the stage 1 and inputs a desired total gain to the gain input unit 33, and then performs control. Laser light is generated from the light source 3 by the device 7.

  The laser light emitted from the light source 3 is reflected by the dichroic mirror 11, deflected by the scanner 13, condensed by the pupil projection lens 15, and the specimen S by the objective lens 19 through the reflection mirror 16 and the imaging lens 17. Is irradiated.

  When fluorescence is generated in the specimen S by being irradiated with the laser light, the fluorescence is collected by the objective lens 19 and passes through the optical path of the laser light via the imaging lens 17, the reflection mirror 16, the pupil projection lens 15, and the scanner 13. Returning, the light passes through the dichroic mirror 11 and is separated from the optical path of the laser light.

  The fluorescence transmitted through the dichroic mirror 11 is collected by the condenser lens 21, and only the fluorescence generated at the focal position of the objective lens 19 in the sample S passes through the pinhole 23. The fluorescence that has passed through the pinhole 23 is converted into parallel light by the collimator lens 25, enters the detection system 30, and is detected by the hybrid detector 31.

  In this case, in the detection system 30, each of the gain setting units 32 </ b> A and 32 </ b> B associated with the total gain input to the gain input unit 33 from the map stored in the information storage unit 35 by the parameter control unit 37. The individual gains are read out, and the applied voltages corresponding to the read individual gains are set in the photocathode gain setting unit 32A and the AD gain setting unit 32B, respectively.

  For example, as shown in FIG. 3, when the total gain 900 is input to the gain input unit 33, the parameter control unit 37 reads the individual gain 900 of the photocathode gain setting unit 32A from the table 1 and The individual gain 1 of the AD gain setting unit 32B is read out.

  Then, the parameter control unit 37 sets the applied voltage −6000 (V) corresponding to the individual gain 900 associated with the total gain 900 according to the table 1 in the photocathode gain setting unit 32A, and the total according to the table 2. The applied voltage 100 (V) corresponding to the individual gain 1 associated with the gain 900 is set in the AD gain setting unit 32B.

  Next, in the hybrid detector 31, the individual gain 900 of the photocathode is set by the photocathode gain setting unit 32A, and the individual gain 1 of the avalanche diode is set by the AD gain setting unit 32B.

  Thereby, the fluorescence is detected by the hybrid detector 31 having the total gain set to 900, converted into a light intensity signal, and sent to the image generation unit 5. Then, the image generator 5 generates an image of the specimen S based on the light intensity signal, and sends it to the monitor 6 for display.

  For example, when the total gain 15000 is input to the gain input unit 33, the parameter control unit 37 reads the individual gain 1500 of the photocathode gain setting unit 32 </ b> A from the table 1 and the AD gain setting unit from the table 2. The individual gain 10 of 32B is read out.

  Then, the parameter control unit 37 sets the applied voltage −8000 (V) corresponding to the individual gain 1500 associated with the total gain 15000 according to the table 1 in the photocathode gain setting unit 32A, and the total according to the table 2 The applied voltage 305 (V) corresponding to the individual gain 10 associated with the gain 15000 is set in the AD gain setting unit 32B.

  Next, in the hybrid detector 31, the individual gain 1500 of the photocathode is set by the photocathode gain setting unit 32A, and the individual gain 10 of the avalanche diode is set by the AD gain setting unit 32B.

  Thereby, the fluorescence is detected by the hybrid detector 31 set to a total gain of 15000, converted into a light intensity signal, and sent to the image generation unit 5. Then, the image generator 5 generates an image of the specimen S based on the light intensity signal, and sends it to the monitor 6 for display.

  As described above, according to the scanning laser microscope 100 according to the present embodiment, when the user inputs the total gain to the gain input unit 33, the parameter control unit 37 causes the gain setting unit having a high S / N according to the map. By setting the applied voltage corresponding to the individual gain associated with the total gain giving priority to 32A (32B) in each gain setting unit 32A, 32B, the hybrid detector 31 can appropriately apply a plurality of applied voltages. Fluorescence is detected and photoelectrically converted according to the total gain combined with the individual gain, and a light intensity signal having a high S / N can be obtained. Therefore, using the hybrid detector 31 whose total gain depends on a plurality of applied voltages, it is possible to always obtain a highly accurate image only by the user inputting a desired total gain.

  In the present embodiment, the case where 900 or 15000 is input as the total gain has been described as an example, but the same applies to the case where the total gain shown in FIG. 3 is 300, 1500, or 150,000. The numerical values of the total gain, individual gain, and applied voltage in the map shown in FIG. 3 are examples, and the present invention is not limited to these.

This embodiment can be modified as follows.
In the present embodiment, the information storage unit 35 includes the tables 1 and 2 in which the individual gains of the gain setting units 32A and 32B are associated with the applied voltages. As a first modification, As shown in FIG. 4, the individual gain and the applied voltage may be associated with each other based on a predetermined function for each of the gain setting units 32A and 32B.

  In this case, in the information storage unit 35, for example, the photocathode gain setting unit 32A having a high S / N is prioritized over the AD gain setting unit 32B, and each individual gain between the gain setting units 32A and 32B is set to each total gain. It may be assumed that a map associated with is stored.

The parameter control unit 37 calculates an applied voltage corresponding to the individual gain associated with the total gain input to the gain input unit 33 for each of the gain setting units 32A and 32B based on a predetermined function. What is necessary is just to set to each gain setting part 32A, 32B.
By doing in this way, it is not necessary to associate an individual gain and a parameter for each gain setting unit 32A, 32B on the map, and the map can be simplified.

  As a second modification, the parameter control unit 37 changes the total gain set in the hybrid detector 31 in accordance with the ratio between the scanning speed input to the input device 9 and the current scanning speed of the scanner 13. It is good as well.

  In this case, the parameter control unit 37 is input to the gain input unit 33 when the scanning speed of the scanner 13 becomes slow as a result of the ratio between the scanning speed input to the input device 9 and the current scanning speed of the scanner 13. Each individual gain associated with a total gain smaller than the total gain may be read from the map, and an applied voltage corresponding to the read individual gain may be set in each gain setting unit 32A, 32B.

  In this way, when the scanning speed of the scanner 13 becomes slow, the total gain of the hybrid detector 31 can be reduced, and the image can be prevented from being saturated even if the exposure time is long.

  On the other hand, if the scanning speed of the scanner 13 is increased as a result of the ratio between the scanning speed input to the input device 9 and the current scanning speed of the scanner 13, the parameter control section 37 is input to the gain input section 33. Each individual gain associated with a total gain larger than the total gain may be read from the map, and an applied voltage corresponding to the read individual gain may be set in each gain setting unit 32A, 32B.

  By doing so, when the scanning speed of the scanner 13 is increased, the total gain of the hybrid detector 31 is increased, and the brightness of the image can be ensured even if the exposure time is shortened.

[Second Embodiment]
Next, a scanning laser microscope according to the second embodiment of the present invention will be described.
As shown in FIG. 5, the scanning laser microscope 200 according to the present embodiment is different from the first embodiment in that the detection system 30 includes an information storage unit 135 that replaces the information storage unit 35 and a calculation unit 139. .
In the following, portions having the same configuration as those of the scanning laser microscope 100 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

  The information storage unit 135 includes priority information (information 1) between the photocathode gain setting unit 32A and the AD gain setting unit 32B, and individual gains that can be set by the photocathode gain setting unit 32A and the AD gain setting unit 32B. Settable range information (information 2) and relational expression information (information 3) for associating individual gains of the photocathode gain setting unit 32A and AD gain setting unit 32B with applied voltages (parameters) are stored. .

In this embodiment, for example, the information storage unit 135 stores information 1, information 2, and information 3 as exemplified below.
That is, as information 1, it is stored that the photocathode gain setting unit 32A is used with priority.
As information 2, the settable range of the individual gain _{Y 1} of the AD gain setting unit 32B:
10 ≦ Y ₁ ≦ 100 (applied voltage conversion: 300 (V) ≦ X1 ≦ 400 (V))
When, the individual gain Y ₂ of the settable range of the photocathode gain setting unit 32A:
500 ≦ Y ₂ ≦ 1500 (applied voltage conversion: −4000 (V) ≦ X2 ≦ −8500 (V))
Is remembered. Here, a, b, and c are constants, and X1 and X2 are applied voltages (V).

Further, as the information 3, AD gain equation associating the individual gain _{Y 1} and applied voltage X1 of AD gain setting unit _32B: Y 1 = a ^{ae x1,} individual gain _{Y 2} and the applied voltage of the photocathode gain setting unit 32A X2 Are stored as follows: Y ₂ = b (X2−c).
Further, the information storage unit 135 stores a total gain: Y ₃ = Y ₁ × Y ₂ .

The calculation unit 139 is input by the gain input unit 33 based on the priority order information (information 1), the settable range information (information 2), and the relational expression information (information 3) stored in the information storage unit 135. together combine individual gain Y ₁ individual gain Y ₂ and AD gain setting section 32B of the photocathode gain setting unit 32A so as to satisfy the total gain, the applied voltage X1 of the combined individual gain Y ₁ corresponding to the individual gain Y ₂ The applied voltage X2 is calculated.

The parameter control unit 37 sets the applied voltages X1 and X2 calculated by the calculation unit 139 in the photocathode gain setting unit 32A and the AD gain setting unit 32B.
The gain input unit 33 is configured to send the total gain input by the user to the calculation unit 139.

The operation of the scanning laser microscope 200 configured as described above will be described.
In the detection system 30, based on the information 1, information 2, and information 3 stored in the information storage unit 135 so as to satisfy the total gain Y ₃ input by the gain input unit 33 by the calculation unit 139, Thus, the applied voltages of the photocathode gain setting unit 32A and the AD gain setting unit 32B are calculated.

That is, in the above setting example, the photoelectron gain setting unit 32A is prioritized by the calculation unit 139. For example, when the total gain Y ₃ input by the gain input unit 33 is smaller than 15000, the calculation unit 139 causes the minimum value of the individual gain Y ₁ of the AD gain setting unit 32B, that is, Y ₁ = 10, individual gain _Y 2 _{= Y} 3 / _{Y 1} photocathode gain setting unit 32A, i.e. are combined and _Y 2 _{= Y} 3/10.

Then, the arithmetic unit 139, the photocathode gain _{equation: Y 2 = b (X2-} c) from the formula, the individual gain of the photocathode gain setting unit 32A _Y 2 _{= Y} 3/10 corresponding to the applied voltage X2, That is, X2 = (1 / 10b) + c is calculated. Also, the calculation unit 139 calculates an applied voltage X1 corresponding to the individual gain Y ₁ = 10 of the AD gain setting unit 32B, that is, X1 = 300, from the AD gain calculation formula: Y ₁ = ae ^x1 .

  Then, the parameter control unit 37 sets the applied voltage X2 = (1 / 10b) + c in the photocathode gain setting unit 32A and sets the applied voltage X1 = 300 in the AD gain setting unit 32B.

On the other hand, if the total gain _{Y 3} which is input by the gain input unit 33 is 15000 or more, the calculation unit 139, the maximum value of the individual gain _{Y 2} photocathode gain setting unit 32A, that is, _Y 2 = 1500, AD gain individual gain _Y 1 _{= Y} 3 / _{Y 2} of the setting unit 32B, that is, combined with _Y 1 _{= Y} 3/1500.

Next, the calculation unit 139 calculates the applied voltage X2 = −8500 corresponding to the individual gain Y ₂ = 1500 of the photocathode gain setting unit 32A from the formula of the photocathode gain calculation formula: Y ₂ = b (X2-c). Calculated. Further, the calculation unit 139 calculates the applied voltage X1 corresponding to the individual gain Y ₁ = Y ₃ / Y ₂ of the AD gain setting unit 32B from the AD gain calculation formula: Y ₁ = ae ^x1 , that is, X1 = log { Y3 / (1500 × a)} is calculated.

  Then, the parameter control unit 37 sets the applied voltage X2 = −8500 in the photocathode gain setting unit 32A, and sets the applied voltage X1 = log {Y3 / (1500 × a)} in the AD gain setting unit 32B. The

Therefore, according to the scanning laser microscope 200 according to the present embodiment, priority order information of the photocathode gain setting unit 32A and AD gain setting unit 32B, and individual gains that can be set for each of these gain setting units 32A and 32B. If the information storage unit 135 stores relational expression information that associates the settable range information and the individual gains for each of the gain setting units 32A and 32B with the applied voltage, the total gain can be input by the gain input unit 33. The parameter control unit 37 gives priority to the desired photocathode gain setting unit 32A (or the AD gain setting unit 32B), and gives each photocathode gain setting unit 32A and AD gain setting unit 32B a combination of individual gains that satisfy the total gain. An applied voltage corresponding to the individual gain can be set.
In this embodiment, the photocathode gain setting unit 32A is given priority as the information 1, but the AD gain setting unit 32B may be given priority. Moreover, the numerical values set in the information 2 and 3 are examples, and are not limited thereto.

  As mentioned above, although each embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes design changes and the like without departing from the gist of the present invention. . For example, the present invention is not limited to those applied to the above-described embodiments and modifications, and may be applied to embodiments that appropriately combine these embodiments and modifications, and is not particularly limited. Moreover, although the photocathode gain setting unit 32A and the AD gain setting unit 32B have been exemplified and described as the gain setting unit, the gain setting unit may be provided with a plurality of gain setting units, and is not limited thereto.

3 Light source
5 Image generation unit 7 Control device (scanning speed setting unit)
9 Input device (scanning speed input part)
13 Scanner (scanning unit)
19 Objective lens 32A Photocathode gain setting section (gain setting section)
32B AD gain setting part (gain setting part)
33 Gain input section 35 Information storage section (map storage section)
37 Parameter control unit 100, 200 Scanning laser microscope 135 Information storage unit 139 Calculation unit S Sample

Claims (6)

A scanning unit that two-dimensionally scans a sample with laser light emitted from a light source;
An objective lens that irradiates the sample with laser light scanned by the scanning unit;
In accordance with a total gain that depends on a plurality of parameters, a light detection unit that detects return light returning from the sample irradiated with laser light and outputs a light intensity signal corresponding to the brightness of the return light;
An image generation unit that converts the light intensity signal output from the light detection unit into luminance information for each pixel corresponding to the scanning position of the scanning unit, and generates an image of the sample;
A gain input unit for the user to input the desired total gain;
A plurality of gain setting units for setting individual gains of the respective parameters in the light detection unit;
A map in which the individual gain and the parameter are associated with each gain setting unit, and the individual gains of the gain setting units are associated with the total gain according to a predetermined priority between the gain setting units. A map storage unit for storing
The individual gain of each gain setting unit associated with the total gain input by the gain input unit is read from the map, and the parameter corresponding to the read individual gain is set in each gain setting unit. A scanning laser microscope comprising a parameter control unit. A scanning unit that two-dimensionally scans a sample with laser light emitted from a light source;
An objective lens that irradiates the sample with laser light scanned by the scanning unit;
In accordance with a total gain that depends on a plurality of parameters, a light detection unit that detects return light returning from the sample irradiated with laser light and outputs a light intensity signal corresponding to the brightness of the return light;
An image generation unit that converts the light intensity signal output from the light detection unit into luminance information for each pixel corresponding to the scanning position of the scanning unit, and generates an image of the sample;
A gain input unit for the user to input the desired total gain;
A gain setting unit for setting an individual gain of each parameter in the light detection unit;
A map storage unit that stores a map in which each individual gain of the gain setting units is associated with each total gain according to a predetermined priority order between the gain setting units;
The individual gain of each gain setting unit associated with the total gain input by the gain input unit is read from the map, and the parameter corresponding to the read individual gain is set in each gain setting unit. A parameter control unit,
A scanning laser microscope in which the individual gain and the parameter are associated with each other based on a predetermined function for each gain setting unit. A scanning speed input unit for a user to input a scanning speed of the scanning unit;
A scanning speed setting section for setting the scanning speed input by the scanning speed input section in the scanning section,
When the scanning speed of the scanning unit decreases according to the ratio of the scanning speed input to the scanning speed input unit and the current scanning speed of the scanning unit, the parameter control unit When each individual gain is read from the map based on a total gain smaller than the input total gain and the scanning speed of the scanning unit is increased, the total gain larger than the total gain input to the gain input unit The scanning laser microscope according to claim 1, wherein each individual gain is read out from the map based on the map. A scanning unit that two-dimensionally scans a sample with laser light emitted from a light source;
An objective lens that irradiates the sample with laser light scanned by the scanning unit;
In accordance with a total gain that depends on a plurality of parameters, a light detection unit that detects return light returning from the sample irradiated with laser light and outputs a light intensity signal corresponding to the brightness of the return light;
An image generation unit that converts the light intensity signal output from the light detection unit into luminance information for each pixel corresponding to the scanning position of the scanning unit, and generates an image of the sample;
A gain input unit for the user to input the desired total gain;
A plurality of gain setting units for setting individual gains of the respective parameters in the light detection unit;
Priority information between these gain setting units, individual gain setting range information that can be set for each gain setting unit, and relational expression information that associates the individual gain and the parameter for each gain setting unit are stored An information storage unit to
Based on the priority order information, the settable range information, and the relational expression information stored in the information storage unit, the individual gain setting units of the individual gain setting units satisfy the total gain input by the gain input unit. A calculation unit for combining the gains and calculating the parameters corresponding to the combined individual gains;
A scanning laser microscope comprising: a parameter control unit configured to set the parameter calculated by the calculation unit in each gain setting unit.   The scanning laser microscope according to claim 1, wherein the predetermined priority is given priority to the gain setting unit having a high S / N.   6. The scanning laser according to claim 1, wherein the light detection unit is a hybrid detector, and a voltage applied to a photocathode of the hybrid detector and a voltage applied to a semiconductor element are respectively used as the parameters. microscope.

Images