JP6205198B2 - Microscope system - Google Patents

Description

  The present invention relates to a microscope system.

  Conventionally, as a detector used in a scanning laser microscope, a GaAsP (Gallium Arsenide Phosphide) compound is used on a photocathode in addition to a multialkali PMT (Photomultiplier Tube) using a multialkali photocathode. GaAsP-PMT and HPD (Hybrid Photo-Detector), which are more sensitive than multi-alkali PMTs, are known.

  GaAsP-PMT and HPD have a significant deterioration in the lifetime of the photocathode compared to the multi-alkali PMT and are very expensive. Therefore, switching between the multi-alkali PMT and the GaAsP-PMT or HPD depending on the importance of observation and measurement. However, it is desirable to suppress deterioration of the photocathode of GaAsP-PMT and HPD.

  However, since the multi-alkali PMT and the GaAsP-PMT and HPD have different HV (High Voltage, applied voltage) gain due to the difference in the multiplication stage structure, it is necessary to adjust the sensitivity after switching, and the GaAsP photoelectric after switching. In order to avoid the deterioration of the surface, the state change of the live specimen or the loss of activity, the sensitivity adjustment needs to be easy and quick.

  Here, a technique for correcting variations in sensitivity among a plurality of fluorescence detectors is known (see, for example, Patent Document 1 and Patent Document 2). The technique described in Patent Document 1 calibrates the sensitivity of a detector corresponding to each wavelength region of fluorescence in a fluorescence spectrometer, and the technique described in Patent Document 2 is a sensitivity between cells in a multi-cell type PMT. Correct.

JP 10-507828 Gazette JP 2006-58237 A

  However, although the techniques described in Patent Documents 1 and 2 can correct variations in sensitivity between fluorescent detectors of the same type or model, Patent Documents 1 and 2 refer to sensitivity correction between different types of PMTs. It has not been. For this reason, even if the techniques disclosed in Patent Documents 1 and 2 are employed, there is a problem that the sensitivity cannot be easily and quickly adjusted when the multi-alkali PMT is switched to GaAsP-PMT or HPD.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a microscope system that can easily and quickly adjust sensitivity associated with switching to a highly sensitive detector.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a microscope main body that irradiates a sample with light and collects observation light emitted from the sample, a first detector that receives and photoelectrically converts the observation light collected by the microscope main body, the first detector is a detector of different, more sensitive than the first detector, a second detector capable of photoelectric conversion by receiving the observation light collected by the microscope main body, wherein A switching unit that switches between the first detector and the second detector as a detector for entering observation light , and when the switching unit switches from the first detector to the second detector, the switching unit immediately before switching. A sensitivity setting unit configured to set an applied voltage to be applied to the second detector based on an applied voltage of the first detector and a ratio of the sensitivity of the second detector to the sensitivity of the first detector; A microscope system is provided.

  According to the present invention, the observation light from the sample is collected by the microscope main body, the observation light is received by the first detector or the second detector, and photoelectric conversion is performed, thereby obtaining image information of the sample. it can. In addition, the second detector having high sensitivity can obtain clearer image information than the first detector.

  In this case, since the first detector and the second detector have different sensitivities, when switching from the first detector to the second detector, image information having the same luminance can be obtained by applying the same voltage. I can't. On the other hand, the applied voltage applied to the second detector is set by the sensitivity setting unit based on the applied voltage of the first detector immediately before switching and the ratio of the sensitivity of the second detector to the sensitivity of the first detector. As a result, the luminance level can be kept substantially constant before and after the switching, and the sensitivity adjustment work associated with the switching can be facilitated and speeded up compared to the case where the applied voltage is readjusted after the switching. .

  As a result, even when the photocathode of the second detector is likely to deteriorate, the deterioration of the photocathode of the second detector after switching is kept to a minimum, and the state change and loss of activity of the living specimen are suppressed. Can do.

  In the above invention, the sensitivity setting unit is further applied to the second detector based on a ratio of a cathode sensitivity characteristic of the photocathode of the second detector to a cathode sensitivity characteristic of the photocathode of the first detector. The applied voltage may be set.

  Since the wavelength dependence of the cathode sensitivity varies depending on the type of the photocathode, this configuration makes it possible to set a more appropriate applied voltage with the cathode sensitivity characteristic corresponding to the wavelength of the observation light actually measured. .

In the above invention, the sensitivity setting unit is further applied to the second detector based on the ratio of the gain characteristic of the multiplication stage of the second detector to the gain characteristic of the multiplication stage of the first detector. The applied voltage may be set.
By configuring in this way, it is possible to set a more appropriate applied voltage even when the configuration of the multiplication stage is different between the first detector and the second detector.

  In the above invention, the first amplification unit that amplifies the photoelectric signal obtained by photoelectric conversion by the first detector, and the second amplification that amplifies the photoelectric signal obtained by photoelectric conversion by the second detector. And when the switching unit switches from the first detector to the second detector, the sensitivity setting unit sets the gain characteristic of the second amplification unit to the ratio of the gain characteristic of the first amplification unit. Based on this, the amplification gain of the second amplifying unit may be set.

  With this configuration, even when the change in the brightness of the image due to the switching of the detector exceeds the adjustment range of the applied voltage applied to the second detector, the magnitude of the luminance before and after the switching. Can be kept almost constant.

In the above invention, the second detector may be a photomultiplier tube or a hybrid photodetector using a GaAsP compound on the photocathode.
Photomultiplier tubes and hybrid photodetectors using a GaAsP compound on the photocathode are more susceptible to degradation of the photocathode and are more expensive, but are superior in conversion efficiency of the photocathode than photomultiplier tubes using a multi-alkali photocathode.

In the above invention, the second detector may detect the intensity of the observation light by a photon counting method.
By comprising in this way, even if observation light is weak, it can detect with a high precision by a 2nd detector.

  According to the present invention, there is an effect that it is possible to easily and quickly perform sensitivity adjustment work associated with switching to a highly sensitive detector.

It is a schematic structure figure showing a microscope system concerning one embodiment of the present invention. It is a schematic block diagram of the system control PC of FIG. It is the figure which looked at the scanning fluorescence confocal microscope apparatus of FIG. 1 from the front. It is the figure which looked at the scanning fluorescence confocal microscope apparatus of FIG. 3 from the side. It is the figure which looked at the scanning fluorescence confocal microscope apparatus of FIG. 3 from upper direction. It is a graph which shows an example of the characteristic numerical value of the multi-alkali PMT and GaAsP-PMT in the microscope system which concerns on the 4th modification of one Embodiment of this invention. It is a graph which shows an example of the HV conversion table in the microscope system which concerns on the 4th modification of one Embodiment of this invention. It is a graph which shows an example of the characteristic numerical value of the multi-alkali PMT and GaAsP-PMT in the microscope system which concerns on the 5th modification of one Embodiment of this invention. It is a graph which shows an example of the HV conversion table in the microscope system which concerns on the 5th modification of one Embodiment of this invention. It is a graph which shows the correlation of HV of the multi alkali PMT and GaAsP-PMT in the microscope system which concerns on the 5th modification of one Embodiment of this invention.

A microscope system according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIGS. 1 to 5, the microscope system 100 according to the present embodiment includes a scanning fluorescent confocal microscope apparatus 1 and a system control PC (Personal Computer) that controls the scanning fluorescent confocal microscope apparatus 1. 3 is provided.

  The scanning fluorescence confocal microscope apparatus 1 irradiates a sample S with excitation laser light (hereinafter referred to as excitation light) and collects fluorescence (observation light) emitted from the sample S, and the microscope body 10. The first detection unit 20 and the second detection unit 30 that detect the fluorescence collected by the light source and acquire a light intensity signal, and an optical path switching mirror (switching unit) that switches between the first detection unit 20 and the second detection unit 30 40.

  The microscope body 10 includes a stage 5 on which the sample S is placed (see FIGS. 3 to 5), a light source (not shown) that generates excitation light, and a dichroic mirror (DM) 11 that reflects the excitation light emitted from the light source. A scanner 13 that scans the excitation light reflected by the dichroic mirror 11, a pupil projection lens 15 that collects the excitation light scanned by the scanner 13, and a parallel light that the excitation light collected by the pupil projection lens 15 And an objective lens 19 that irradiates the sample S with the excitation light converted into parallel light by the imaging lens 17 and condenses the fluorescence generated in the sample S.

  The dichroic mirror 11 reflects the excitation light from the light source, while allowing the fluorescence generated in the sample S, collected by the objective lens 19 and returning to the optical path of the excitation light to pass therethrough, and branches the fluorescence from the optical path of the excitation light. It is like that.

  The scanner 13 is accommodated in a scanning unit (SU) 7 shown in FIGS. The scanner 13 is, for example, a two-axis galvanometer mirror, and is configured by opposing two galvanometer mirrors (not shown) that can swing around mutually orthogonal axes. The scanner 13 can swing the two galvanometer mirrors to deflect the excitation light and scan the excitation light two-dimensionally (X-axis direction and Y-axis direction) on the sample. .

  The first detection unit 20 is built in the scanning unit 7. The first detection unit 20 includes a multi-alkali PMT (Photo Multiplier Tube, photomultiplier tube, first detector) 21 that detects fluorescence and outputs a light intensity signal having a magnitude corresponding to the amount of light, and a multi-alkali PMT 21. A first HV power supply 23 that applies HV (High Voltage, applied voltage) and a first amplifier (first amplification unit) 25 that amplifies the light intensity signal output from the multi-alkali PMT 21 are provided.

  The multi-alkali PMT 21 is a side-on type PMT and includes a multi-alkali photocathode (not shown) that receives fluorescence and photoelectrically converts it into a light intensity signal.

  The 2nd detection unit 30 is accommodated in the high sensitivity detection unit (HSD: High Sensitivity Detectors) 9 shown in FIGS. The second detection unit 30 detects fluorescence, performs photoelectric conversion, and outputs a light intensity signal having a magnitude corresponding to the amount of light, and a GaAsP-PMT (Gallium Arsenide Phosphide-PMT, second detector) 31, GaAsP A second HV power supply 33 that applies HV to the PMT 31 and a second amplifier (second amplification unit) 35 that amplifies the light intensity signal output from the GaAsP-PMT 31 are provided.

  The GaAsP-PMT 31 is a head-on type PMT and includes a photocathode (not shown) using a GaAsP compound. This GaAsP-PMT31 has higher sensitivity than the multi-alkali PMT21.

  The optical path switching mirror 40 is provided so as to be able to be inserted into and removed from the fluorescent optical path that has passed through the dichroic mirror 11. The optical path switching mirror 40 reflects fluorescence toward the first detection unit 20 when inserted in the fluorescence optical path. When the optical path switching mirror 40 is removed from the optical path of fluorescence, the fluorescence is incident on the second detection unit 30 as it is.

  As shown in FIG. 2, the system control PC 3 includes a control main body unit (sensitivity setting unit) 41 that performs apparatus setting, image acquisition, image analysis, and the like, and an input unit 43 through which a user inputs instructions to the control main body unit 41. And a monitor 45 for displaying an image of the sample S and the like.

  The control main body 41 sets the scanning condition of the scanner 13, the switching of the optical path switching mirror 40, the setting of the amplification gains of the first amplifier 25 and the second amplifier 35, and the first HV in accordance with the user instruction input to the input unit 43. HV settings for the power source 23 and the second HV power source 33 are performed.

  In addition, the control main body 41 controls the operation of the scanner 13 and converts the light intensity signal sent from the first amplifier 25 or the second amplifier 35 into luminance information for each pixel corresponding to the scanning position of the scanner 13. The image information of the sample S is generated. The control main body 41 displays the generated image information on the monitor 45.

  The control main body 41 can read the generated image information, perform arithmetic processing, and analyze the image. By performing apparatus setting, image acquisition, image analysis, and the like by one control main body 41, it is possible to comprehensively refer to, compare, and calculate HV and amplification gain setting values and image brightness as internal data. it can.

  Further, when the control main body 41 switches from the multi-alkali PMT 21 to the GaAsP-PMT 31 by the optical path switching mirror 40, the control main body 41 sets the HV of the multi-alkali PMT 21 and the HV gain of the multi-alkali PMT 21 set in the first HV power supply 23 immediately before switching. Based on the ratio of the HV gain (sensitivity) of the GaAsP-PMT 31 to (sensitivity), the initial value of HV applied to the GaAsP-PMT 31 is set in the second HV power supply 33.

  For example, the control main body 41 calculates the initial value of HV so that the image obtained after switching to the GaAsP-PMT 31 has substantially the same brightness as the image obtained by the multi-alkali PMT 21 before switching. After the initial value of HV is set in the second HV power supply 33, the multi-alkali PMT 21 is switched to the GaAsP-PMT 31.

  The HV gain characteristics of the multi-alkali PMT 21 and the HV gain characteristics of the GaAsP-PMT 31 can be obtained as typical data by inquiring these PMT manufacturers. It can also be acquired as an actual measurement value during the manufacturing process of the PMT or after the user installs the PMT.

Here, a method for calculating the ratio of the HV gain of the GaAsP-PMT 31 to the HV gain of the multi-alkali PMT 21 will be described.
As a specification example of the multi-alkali PMT 21, for example, the number of multiplication stages (dynodes): 9, applied voltage between the anode and the cathode: 1000 V, cathode radiation sensitivity: 70 mA / W, gain: 9.5 × 10 ⁶ , anode radiation Sensitivity: 665 × 10 ³ A / W. The index k determined by the electrode structure / material of the multiplication stage is set to 0.75.

In this case, since the anode radiation sensitivity and (HV) ^{k · n} are in a proportional relation, the calculation formula of anode radiation sensitivity = 3.74 × 10 ⁻¹⁵ × (HV) ^6.75 is used. Anode radiation sensitivity can be correlated.

On the other hand, as a specification example of GaAsP-PMT31, for example, the number of multiplication stages: 9, applied voltage between anode and cathode: 800 V, cathode radiation sensitivity: 200 mA / W, gain: 3 × 10 ⁶ , anode radiation sensitivity: 600 × 10 ³ A / W. The index k is set to 0.75.

In this case, similarly to the multi-alkali PMT 21, the anode radiation sensitivity and (HV) ^{k · n} are in a proportional relationship, and therefore the anode radiation sensitivity = 1.52 × 10 ⁻¹⁴ × (HV) ^6.75 Thus, HV of GaAsP-PMT31 and anode radiation sensitivity can be correlated.

Then, the voltage applied to multialkali PMT21 and _{HV ma,} the initial value of the voltage applied to the GaAsP-PMT31 and _{HV gap,} the anode radiation sensitivity and GaAsP-PMT31 when the multi-alkali PMT21 used in _{HV ma HV gap} Assuming that the anode irradiation sensitivity is the same as that used in the above, the following calculation formula (1) is established.
Anode radiation sensitivity = 3.74 × 10 ⁻¹⁵ × (HV _ma ) ^6.75 = 1.52 × 10 ⁻¹⁴ × (HV _gap ) ^6.75 (1)

From the calculation formula (1), a relational expression of HV _gap = 0.812 × HV _ma is obtained.
Therefore, in this specification example, the ratio of the HV gain of the GaAsP-PMT 31 to the HV gain of the multi-alkali PMT 21 is 0.812.

The operation of the microscope system 100 configured as described above will be described.
When observing the sample S using the multi-alkali PMT 21 by the microscope system 100 according to the present embodiment, the control main body 41 inserts the optical path switching mirror 40 in the fluorescence optical path and sets the HV _ma in the first HV power supply 23. . Then, the sample S is placed on the stage 5 and excitation light is generated from the light source.

  The excitation light emitted from the light source is reflected by the dichroic mirror 11, deflected by the scanner 13, condensed by the pupil projection lens 15, converted into parallel light by the imaging lens 17, and converted into parallel light by the objective lens 19. Is irradiated. Thereby, the excitation light is scanned two-dimensionally on the sample S according to the swinging motion of the scanner 13.

  When fluorescence is generated in the sample S by irradiation with the excitation light, the fluorescence is collected by the objective lens 19 and returns to the optical path of the excitation light via the imaging lens 17, the pupil projection lens 15, and the scanner 13, and becomes dichroic. The light passes through the mirror 11 and is branched from the optical path of the excitation light. The fluorescence transmitted through the dichroic mirror 11 is reflected by the optical path switching mirror 40 and enters the first detection unit 20.

  In the first detection unit 20, fluorescence is detected by the multi-alkali PMT 21, and a light intensity signal having a magnitude corresponding to the amount of light is output. The light intensity signal is amplified by the first amplifier 25, and the system control PC 3 It is sent to the control main body 41.

  In the control main body 41, the incident light intensity signal is converted into luminance information for each pixel corresponding to the scanning position of the scanner 13, and image information of the sample S is generated. The generated image information is displayed on the monitor 45. Thereby, the sample S can be observed with the image obtained by the multi-alkali PMT 21.

Next, when the sample S is observed using the GaAsP-PMT 31, the control main body 41 removes the optical path switching mirror 40 from the fluorescent light path and sets the HV _gap in the second HV power supply 33. Then, similarly to the case where the multi-alkali PMT 21 is used, the microscope body 10 irradiates the sample S with excitation light.

  In this case, the fluorescence generated in the sample S, condensed by the objective lens 19 and returning the optical path of the excitation light passes through the dichroic mirror 11 and enters the second detection unit 30 without passing through the optical path switching mirror 40.

  In the second detection unit 30, fluorescence is detected by the GaAsP-PMT 31 and a light intensity signal is output, and the light intensity signal is amplified by the second amplifier 35 and sent to the control main body 41. Then, the control main body 41 generates image information of the sample S based on the light intensity signal and displays it on the monitor 45. Thereby, the sample S can be observed with a clearer image obtained by the GaAsP-PMT31 having higher sensitivity than the multi-alkali PMT21.

  Here, the GaAsP-PMT 31 is more susceptible to deterioration of the photocathode and more expensive than the multi-alkali PMT 21. Therefore, for example, in the preliminary experiment at the stage of optimizing various experimental conditions, the multi-alkali PMT 21 is used, and only the main experiment is switched to the GaAsP-PMT 31 and used to suppress deterioration of the photoelectric surface of the GaAsP-PMT 31. It is desirable to do.

Hereinafter, a case where a preliminary experiment is performed using the multi-alkali PMT 21 and the experiment is performed by switching to the GaAsP-PMT 31 will be described.
In this case, the multi-alkali PMT 21 and the GaAsP-PMT 31 have different HV gains (sensitivities) due to the difference in the multiplication stage structure. Therefore, when switching from the multi-alkali PMT 21 to the GaAsP-PMT 31, the same value of HV is applied. Brightness image information cannot be obtained.

For example, the difference between HV _ma and HV _gap is as small as x0.812 (23% difference), but the multiplication gain contributes about four times. Therefore, if the multi-alkali PMT 21 is switched to the GaAsP-PMT 31 without changing the HV setting, the image becomes four times brighter.

  On the other hand, in the microscope system 100 according to the present embodiment, when the control main body 41 switches from the multi-alkali PMT 21 to the GaAsP-PMT 31 by the optical path switching mirror 40, the HV of the multi-alkali PMT 21 and the multi-alkali PMT 21 immediately before switching are switched. The HV applied to the GaAsP-PMT 31 is set based on the ratio of the sensitivity of the GaAsP-PMT 31 to the sensitivity.

That is, in the preliminary experiment, the user adjusts the HV _ma so that a clear image can be obtained by the multi-alkali PMT 21 and an image of the sample S is acquired. Next, in the case of this experiment, the control main body 41 calculates an HV _gap so that the brightness of the images before and after switching from the multi-alkali PMT 21 to the GaAsP-PMT 31 is substantially the same based on the latest HV _ma. The second HV power supply 33 is set and switched to the GaAsP-PMT31.

  Therefore, according to the microscope system 100 according to the present embodiment, the luminance level is kept substantially constant before and after switching from the multi-alkali PMT 21 to the GaAsP-PMT 31, and the HV is readjusted after switching to the GaAsP-PMT 31. Compared to the above, it is possible to facilitate and speed up the sensitivity adjustment work associated with switching. As a result, the deterioration of the photocathode of the GaAsP-PMT 31 after switching can be minimized, and the state change and loss of activity of the living sample S can be suppressed.

  In the present embodiment, when switching to the GaAsP-PMT 31, the user may additionally adjust the HV of the GaAsP-PMT 31 as necessary to optimize the image quality.

In the present embodiment, the control main body 41 may set the amplification gain of the second amplifier 35 based on the ratio of the gain characteristic of the second amplifier 35 to the gain characteristic of the first amplifier 25.
By doing in this way, even if the change in the brightness of the image due to the switching from the multi-alkali PMT 21 to the GaAsP-PMT 31 exceeds the adjustment range of the HV _gap , the magnitude of the luminance before and after the switching. Can be kept almost constant.

This embodiment can be modified as follows.
In the present embodiment, as the specification examples of the multi-alkali PMT 21 and the GaAsP-PMT 31, the multiplication stages are the same (9 stages) and are related in a simple linear proportion. For example, the multi-alkali PMT 21 and the GaAsP-PMT 31 Even if the number of amplification stages is different, the conversion calculation can be executed in the same manner.

In this case, the order is converted according to the multiplication stage number, and the calculation formula becomes slightly complicated. Therefore, as the first modification, HV conversion table obtained by converting the _{HV gap} of _{HV ma} and GaAsP-PMT31 multi alkali PMT21 (LUT: Look-Up- Table, not shown) prepared in advance, the main control unit 41 HV _gap may be set with reference to the HV conversion table.

  In the present embodiment, the GaAsP-PMT 31 is exemplified as the second detector. However, the second modification uses a GaAsP compound on the photocathode as the second detector and an avalanche in the multiplication stage. An HPD (Hybrid Photo-Detector, not shown) using a diode may be employed.

Even when HPD is adopted as the second detector, the sensitivity characteristic obtained by the multi-alkali PMT 21 is similarly calculated by grasping the multiplication characteristic in advance and performing conversion, and the initial value of HV _gap is set. be able to.

Next, as a third modification, the control main body 41 is further applied to the GaAsP-PMT 31 based on the ratio of the cathode sensitivity characteristic of the photocathode of the GaAsP-PMT 31 to the cathode sensitivity characteristic of the photocathode of the multi-alkali PMT 21. The HV _gap to be set may be set.

Since the wavelength dependence of cathode sensitivity usually varies depending on the type of photocathode, PMT desirably derives the above relational expression from the cathode sensitivity characteristics corresponding to the fluorescence wavelength actually measured. In this case, the wavelength characteristics to be referred to may be selected according to the type of fluorescent filter set and used in the apparatus depending on the type of fluorescent dye or fluorescent protein that is fluorescently labeled on the biological sample to be measured.
By doing in this way, more suitable HV _gap can be set with the cathode sensitivity characteristic corresponding to the wavelength of the observation light actually measured.

  The sensitivity gain and the multiplication gain may have a large individual difference for each PMT. In the manufacturing process built in the apparatus to the user delivery process, the calculation / conversion table may be created by referring to the shipping test data attached at the time of delivery from the PMT manufacturer and changing the above specification numerical values for each individual. By doing so, it is possible to perform more accurate conversion without being affected by individual differences in PMTs, saving the user's trouble and shortening the time required for experiment preparation.

Next, as a fourth modification, the control main body 41 further applies to the GaAsP-PMT 31 based on the ratio of the gain characteristic of the multiplication stage of the GaAsP-PMT 31 to the gain characteristic of the multiplication stage of the multi-alkali PMT 21. It is good also as setting HV _gap .

  In the above calculation example, the index k determined by the electrode structure / material of the PMT multiplication stage has been described by exemplifying 0.75 which is a normal intermediate value of 0.7 to 0.8. When the structure of the multiplication stage of GaAsP-PMT31 is different, more accurate conversion can be performed by using an index k corresponding to each structure.

For example, the exponent k may be calculated by actually measuring the multiplication gain by changing HV such as 900V and 600V. Moreover, a standard value measured in advance may be used, or a numerical value obtained by actual measurement for each PMT in consideration of variation may be used.
By doing in this way, when the structure of the multiplication stage of the multi-alkali PMT 21 and the GaAsP-PMT 31 is different, a more appropriate HV _gap can be set.

Here, an example of creating an HV conversion table will be described.
First, the PMT gain is obtained by the following calculation formula (2).
Gain = A × (HV) ^{k · n} (2)

  However, amplifiers (first amplifier 25 and second amplifier 35) applied to the multi-alkali PMT 21 and the GaAsP-PMT 31 are different, and amplifier circuit gains are different. Therefore, it is preferable to use the following calculation formula (3).

Total Gain = Gamp × A × (HV) ^{k · n} (3)
here,
Gamp: Amplifier gain.

An example of characteristic values of the multi-alkali PMT 21 and GaAsP-PMT 31 is shown in FIG.
The HV conversion table shown in FIG. 7 can be created from the calculation formula (3) and the characteristic numerical values shown in FIG. In FIG. 6, it is not necessary to input the circuit gain itself for Gamp (amplifier gain), and the relative relationship between the setting value when using the multi-alkali PMT 21 and the setting value when using the GaAsP-PMT 31 is correctly input. do it.

Even if the type of each photocathode of each of the multi-alkali PMT 21 and the GaAsP-PMT 31 and the structure of each multiplication stage are different, the characteristic values are obtained in advance so that when the multi-alkali PMT 21 is switched to the GaAsP-PMT 31 An HV conversion table can be created by HV conversion so that the brightness does not change, and an initial value of HV _gap applied to the GaAsP-PMT 31 can be set.

  In this modification, the effect of the amplifier gain is taken into account, but in reality, the signal gain is changed by a variety of implementation means such as the integration gain of the analog integration circuit and the data calculation after digital conversion of the analog signal. can do. It goes without saying that the gist of the present invention can be universally applied even if the means for changing the signal gain changes.

Next, in the fourth modification, attention is paid only to the gains of the multi-alkali PMT 21 and GaAsP-PMT 31 and the first amplifier 25 and the second amplifier 35. However, as the fifth modification, the multi-alkali PMT 21 and GaAsP-PMT 31 It is good also as setting HV _gap including the difference in the transmission efficiency in a detection optical system when performing fluorescence detection by switching.

The calculation formula (4) in this case is as follows.
Total Gain = Top × Gamp × A × (HV) ^{k · n} (4)
here,
Topt: Transmission efficiency of the detection optical system.

The transmission optical path of the detection optical system is given by integrating the reflectance and / or transmittance of each optical element such as the optical path switching mirror 40, the dichroic mirror 11 and the barrier filter (not shown) existing in the detection optical path. . Since the purpose is to adjust the HV between the multi-alkali PMT 21 and the GaAsP-PMT 31, it is not always necessary to know the absolute value of the transmittance / reflectance, and if the relative ratio is known, the HV can be calculated. it can.
For example, fluorescence may be detected by introducing fluorescence to an external detector through fiber connection, and HV may be calculated in consideration of transmission efficiency through the fiber.

  In FIG. 1, if the reflectance of the optical path switching mirror 40 is 90%, the top of the multi-alkali PMT 21 is 0.90. On the other hand, since the fluorescence is incident on the GaAsP-PMT 31, the Top is 1.00. Since Top has only a relative ratio in calculation, 90 and 100 may be input in the calculation formula (4), respectively.

  In fluorescence detection, a dichroic mirror (not shown) may be inserted in the detection optical path. In that case, it is good also as calculating as Topt using the spectral wavelength reflectance or spectral wavelength transmittance | permeability corresponding to the fluorescence wavelength to detect. By doing in this way, more effective HV conversion can be performed.

In this modification, the HV conversion table shown in FIG. 9 can be created from the above calculation formula (4) and the characteristic values shown in FIG. The HV correlation between the multi-alkali PMT 21 and the GaAsP-PMT 31 is as shown in FIG. 10, the vertical axis represents the _{HV ma} multi alkaline PMT21, the horizontal axis represents the _{HV gap} of GaAsP-PMT31.

In the HV conversion table shown in FIG. 9, the range of HV set by the multi-alkali PMT 21 is expanded to 500V to 1100V. At this time, the conversion result of HV set in the GaAsP-PMT 31 is 449V to 966V in calculation.
Therefore, it is desirable to provide a limit (upper limit / lower limit) for HV applicable in GaAsP-PMT31.

The upper limit is as follows.
That often maximum rated HV is lower than the multi-alkali PMT21 in GaAsP-PMT31, Assuming that the 900V the upper limit of _{HV gap,} in terms of value if you set the _{HV ma} larger than 1040V in multialkali PMT21 Can not apply.

  In such a case, a warning may be displayed to the user. Further, when the amplifier gain of the second amplifier 35 can be set high, the amplifier gain may be reset to a large setting value and corrected including the amplifier gain.

The lower limit is as follows.
That is, when the HV _gap applied to the GaAsP-PMT 31 is lowered and the GaAsP-PMT 31 is used, the multiplication factor is lowered, so that more electrons are emitted from the photocathode, the deterioration is promoted, and the lifetime is further shortened. Become. For this reason, it is desirable for the GaAsP-PMT 31 to set the lower limit of the HV set value.

Assuming that the lower limit of the HV setting value in the GaAsP-PMT 31 is 500 V, the conversion value cannot be applied when the HV _ma having a numerical value lower than 540 V is set in the multi-alkali PMT 21. In such a case, a warning may be displayed to the user. When the amplifier gain of the second amplifier 35 can be set low, the amplifier gain may be reset to a low setting value and corrected including the amplification gain.

  Next, as a sixth modification, the GaAsP-PMT 31 may detect the intensity of fluorescence by a photon counting method. In this case, the photon count value of GaAsP-PMT 31 corresponding to the HV setting value of the multi-alkali PMT 21 may be calculated and stored in advance.

When the control main body 41 switches from the multi-alkali PMT 21 to the GaAsP-PMT 31 by the optical path switching mirror 40, the HV _ma of the multi-alkali PMT 21 immediately before switching and the HV gain of the GaAsP-PMT 31 with respect to the HV gain of the multi-alkali PMT 21. The photon count value of GaAsP-PMT 31 may be set based on the ratio.
By doing in this way, even if observation light is weak, it can detect with high precision by GaAsP-PMT31.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.

10 Microscope body 21 Multi-alkali PMT (first detector)
25 1st amplifier (1st amplification part)
31 GaAsP-PMT (second detector)
35 Second amplifier (second amplifier)
40 Optical path switching mirror (switching unit)
41 Control body (sensitivity setting part)
100 Microscope system S Sample

Claims (6)

A microscope body that irradiates the sample with light and collects the observation light emitted from the sample;
A first detector capable of receiving and photoelectrically converting observation light collected by the microscope body;
The first detector is a different type of detector, has a higher sensitivity than the first detector , and can receive and photoelectrically convert the observation light collected by the microscope main body, and a second detector;
A switching unit for switching between the first detector and the second detector as a detector for entering the observation light ;
When switching from the first detector to the second detector by the switching unit, the applied voltage of the first detector immediately before switching and the ratio of the sensitivity of the second detector to the sensitivity of the first detector And a sensitivity setting unit that sets an applied voltage to be applied to the second detector.   The sensitivity setting unit further sets an applied voltage to be applied to the second detector based on a ratio of a cathode sensitivity characteristic of the photocathode of the second detector to a cathode sensitivity characteristic of the photocathode of the first detector. The microscope system according to claim 1.   The sensitivity setting unit further sets an applied voltage to be applied to the second detector based on a ratio of a gain characteristic of the multiplication stage of the second detector to a gain characteristic of the multiplication stage of the first detector. The microscope system according to claim 1 or 2. A first amplifying unit for amplifying a photoelectric signal obtained by photoelectric conversion by the first detector;
A second amplification unit for amplifying a photoelectric signal obtained by photoelectric conversion by the second detector;
When switching from the first detector to the second detector by the switching unit, the sensitivity setting unit is based on the ratio of the gain characteristic of the second amplification unit to the gain characteristic of the first amplification unit, The microscope system according to any one of claims 1 to 3, wherein an amplification gain of the second amplification unit is set.   The microscope system according to any one of claims 1 to 4, wherein the second detector is a photomultiplier tube or a hybrid photodetector using a GaAsP compound on a photocathode.   The microscope system according to any one of claims 1 to 5, wherein the second detector detects the intensity of observation light by a photon counting method.

Images