JP2008170973A - Confocal laser microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal laser microscope capable of restraining an increase in time required to form an extended image or three dimensional image. <P>SOLUTION: The confocal laser microscope includes a laser light source 1, an objective lens 5, a specimen table 6A, a secondary scanning mechanism 3, and a Z movement mechanism 1409, a pin hole 8, and an optical detector 9, and formes an extended image or three dimensional image of a specimen 6. If it is determined that at least the detecting sensitivity of the optical detector 9 or the emission power of the laser light source 1 is adjusted in the course of movement of the objective lens 5 or specimen table 6A within a moving distance, luminance detected before or after the adjustment of the detecting sensitivity or emission power is corrected so as to have a relative relation with luminance detected before or after the adjustment of the detecting sensitivity or emission power. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a confocal laser microscope.

FIG. 11 is a diagram showing an existing confocal laser microscope.
A confocal laser microscope shown in FIG. 11 includes a laser light source 1, a PBS (Polarization Beam Splitter) 2, a two-dimensional scanning mechanism 3, a ¼λ plate 4, an objective lens 5, a condenser lens 7, and a pin. A hall 8, a photodetector 9, a detection sensitivity adjustment unit 10, a laser power adjustment DA converter 11, a controller 12, and a monitor 13 are provided.

  In the confocal laser microscope shown in FIG. 11, the light emitted from the laser light source 1 passes through the PBS 2 and then enters the two-dimensional scanning mechanism 3. The two-dimensional scanning mechanism 3 guides the incident light to the objective lens 5 while scanning the two-dimensionally. Since the two-dimensional scanning mechanism 3 is arranged at a position conjugate with the pupil of the objective lens 5, the light incident on the pupil of the objective lens 5 is condensed to scan the sample 6.

  The light reflected from the surface of the sample 6 is again incident on the PBS 2 from the objective lens 5 via the 1 / 4λ plate 4 and the two-dimensional scanning mechanism 3, and then reflected by the PBS 2 toward the condenser lens 7 and collected. The light is condensed on the pinhole 8 by the optical lens 7.

  Of the reflected light collected on the pinhole 8, the light reflected at a point other than the focal point on the sample 6 is cut by the pinhole 8, and only the reflected light that has passed through the pinhole 8 is detected by the photodetector 9. Detected. The photodetector 9 converts the luminance of the detected reflected light into an electrical signal and outputs it to the controller 12 via the detection sensitivity adjustment unit 10.

  The quarter λ plate 4 converts incident light from linearly polarized light to circularly polarized light. The objective lens 5 is movable in the optical axis direction (Z direction) of light emitted from the objective lens 5 by a Z movement mechanism (not shown). The operations of the two-dimensional scanning mechanism 3 and the Z moving mechanism are controlled by the controller 12.

  As an image created by the confocal laser microscope, there are an extended image (an image in which all the concaves and convexes are focused by laminating one tomographic image having a shallow focal depth in the Z direction) and a three-dimensional image. When creating these extended images or three-dimensional images, for example, the user does not have insufficient or saturated (excessive) luminance output from the photodetector 9 to the detection sensitivity adjustment unit 10 with respect to the target luminance range. As described above, the brightness adjustment value displayed next to the confocal image displayed in real time on the monitor 13 is optimally adjusted.

  In addition, during the creation of an extended image or a three-dimensional image, if the luminance output from the photodetector 9 to the detection sensitivity adjustment unit 10 is insufficient or saturated with respect to the target luminance range, the luminance adjustment value is readjusted. The tomographic image is acquired again from the beginning.

In addition, when an extended image or a three-dimensional image of the sample 6 having a plurality of surfaces each having a different reflectance is created, the brightness adjustment value is optimally adjusted in advance for each surface having a different reflectance. (For example, see Patent Document 1)
JP-A-8-292198

However, the above existing confocal laser microscope has the following problems.
That is, there is a problem that the time for creating the extended image and the three-dimensional image increases by the time for acquiring the confocal image in advance and the time for adjusting the luminance adjustment value using the confocal image.

  In addition, when the brightness value is insufficient or saturated during the creation of the extended image or the three-dimensional image, it is necessary to readjust the brightness adjustment value, and the extended image of the sample 6 having a plurality of surfaces having different reflectances. In the case of creating a three-dimensional image, the number of tomographic images for luminance adjustment increases, so the time required for adjusting the luminance increases accordingly, and the time for creating an extended image or a three-dimensional image further increases. is there.

  Accordingly, an object of the present invention is to provide a confocal laser microscope capable of suppressing an increase in the time for creating an extended image or a three-dimensional image.

In order to solve the above problems, the present invention adopts the following configuration.
That is, the confocal laser microscope of the present invention includes a laser light source, an objective lens for condensing the laser light emitted from the laser light source on the sample, and two-dimensional scanning for scanning the laser light two-dimensionally on the sample. Means, a moving means for moving the objective lens or a sample stage on which the sample is placed at a predetermined interval in a movement distance set in advance in the optical axis direction of the light emitted from the objective lens, and the objective lens A confocal stop means disposed at a position conjugate with the condensing position; and a photodetector for detecting the brightness of light that has passed through the confocal stop means, and based on the brightness and the height information of the sample A confocal laser microscope for creating an extended image or a three-dimensional image of the sample, wherein the detection sensitivity or the level of the photodetector is set so that the luminance falls within a target luminance range. An adjusting means for adjusting at least one of the output power of the light source, and at least one of the detection sensitivity or the output power by the adjusting means while the objective lens or the sample stage moves within the moving distance. When the detection sensitivity or the output power is adjusted, the brightness detected before or after the detection sensitivity or the output power is adjusted is correlated with the brightness detected before or after the detection sensitivity or the output power is adjusted. Correlating means for correcting so as to be.

  The correlating means may calculate a ratio between the luminance detected before the detection sensitivity or the output power is adjusted and the luminance detected after the detection sensitivity or the output power is adjusted. A value obtained by multiplying the luminance detected before or after the emission power is adjusted is set as a value after correction of the luminance detected before or after the detection sensitivity or the emission power is adjusted. May be.

  Further, the correlation means may be configured to determine whether at least one of the detection sensitivity or the output power is adjusted every time the objective lens or the sample stage moves at the predetermined interval. .

  Further, the correlation means may be configured to detect the detection sensitivity or the output power while the objective lens or the sample stage is moving within the movement distance after the movement of the objective lens or the sample stage is completed. It may be configured to determine whether at least one of the adjustments has been made.

  The photodetector may be a photomultiplier or a photodiode, and the adjusting means may be configured to adjust the detection sensitivity by controlling a voltage applied to the photodetector.

  The adjusting unit may be configured to adjust the emission power by controlling a driving current to the laser light source.

  According to the present invention, it is possible to suppress an increase in the time for creating an extended image or a three-dimensional image.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
<First Embodiment>
FIG. 1 is a diagram showing a confocal laser microscope according to the first embodiment of the present invention. In addition, the same code | symbol is attached | subjected to the same structure as the structure shown in FIG.

  The confocal laser microscope shown in FIG. 1 includes a laser light source 1, a PBS 2, a two-dimensional scanning mechanism 3, a ¼λ plate 4, an objective lens 5, a condenser lens 7, a pinhole 8, and light detection. A detector 9, a detection sensitivity adjustment unit 10, a DA converter 11 for laser power adjustment, a controller 12, and a monitor 13. A Z moving mechanism 1409 (to be described later) includes a motor or objective lens for moving the objective lens 5 or the sample stage 6A on which the specimen 6 is placed in the optical axis direction (Z direction) of the light emitted from the objective lens 5. 5 or an AD converter or the like for converting a signal indicating the current position of the sample stage 6A from an analog signal to a digital signal.

FIG. 2 is a diagram showing the detection sensitivity adjustment unit 10.
The detection sensitivity adjustment unit 10 shown in FIG. 2 includes a high voltage power supply circuit 14, an amplifier 15, an AD converter 16, and a DA converter 17.

The DA converter 17 converts the luminance adjustment value output from the controller 12 from a digital signal to an analog signal and outputs the analog signal to the high voltage power supply circuit 14.
The high-voltage power supply circuit 14 applies an applied voltage HV based on the brightness adjustment value output from the DA converter 17 to the photodetector 9. The photodetector 9 adjusts the detection sensitivity based on the applied voltage HV that is applied.

The amplifier 15 amplifies the signal detected by the photodetector 9 and indicating the luminance of the reflected light that has passed through the pinhole 8.
The AD converter 16 converts the signal amplified by the amplifier 15 from an analog signal to a digital signal and outputs the signal to the controller 12.

FIG. 3 is a diagram illustrating the luminance value correlation adjusting unit 20 provided in the controller 12.
The luminance value correlation adjustment unit 20 shown in FIG. 3 includes a luminance value storage unit 1401, a height information storage unit 1402, a luminance value correlation calculation unit 1403, a luminance adjustment value calculation unit 1404, a luminance adjustment I / O 1405, A luminance adjustment value storage unit 1406, a Z movement mechanism I / O 1407, a Z movement mechanism driver 1408, and an image transfer I / O 1410 are provided.

  In the confocal laser microscope shown in FIG. 1, similarly to the confocal laser microscope shown in FIG. 11, the condensing position by the objective lens 5 and the position of the pinhole 8 are in an optically conjugate positional relationship. That is, when the sample 6 is at a position where light is collected by the objective lens 5, the reflected light from the sample 6 is collected on the pinhole 8 and passes through the pinhole 8. When the sample 6 is at a position deviated from the condensing position by the objective lens 5, the reflected light from the sample 6 does not collect on the pinhole 8 and therefore does not pass through the pinhole 8. Accordingly, the relationship between the relative position of the objective lens 5 and the sample 6 at this time and the output of the photodetector 9 is as follows. That is, when the sample 6 is at the condensing position of the objective lens 5, the luminance value detected by the photodetector 9 is maximized. Then, as the relative position between the objective lens 5 and the sample 6 increases from this position, the output of the photodetector 9 decreases rapidly. Therefore, the condensing position by the objective lens 5 is two-dimensionally scanned by the two-dimensional scanning mechanism 3, and the luminance value detected by the photodetector 9 at that time is used as the scanning position information in the xy plane direction in the two-dimensional scanning mechanism 3. When the image is associated and imaged by the controller 12 and the image is displayed on the monitor 13, a confocal image representing only a specific height of the sample 6 is obtained. Further, the luminance value detected by the photodetector 9 is acquired in the same manner as described above while moving the objective lens 5 or the sample stage 6A in the Z direction by the Z moving mechanism 1409. Then, the luminance value having the largest luminance value among all the luminance values in the pixels at the same position in the xy plane direction and the height information of the objective lens 5 corresponding to the luminance value are all in the xy plane direction. An extended image or a three-dimensional image is created using the number of pixels.

Next, a more specific process for creating an extended image or a three-dimensional image will be described.
First, while viewing the confocal image of the sample 6 displayed on the monitor 13, the user operates the Z moving mechanism 1409 on the operation screen displayed next to the confocal image to move the objective lens 5 in the Z direction. The movement distance of the objective lens 5 is set in advance. The movement distance is stored in a predetermined memory of the controller 12, for example.

  Next, when the setting of the movement distance is completed, the objective lens 5 rises within the movement distance at a predetermined movement pitch ΔZ, and the luminance value of all pixels in the xy plane direction for each movement pitch ΔZ. I is acquired. That is, when the luminance values I for all the pixels in the xy plane direction are obtained in certain height information Z (k), the luminance values I are associated with the height information Z (k) in the luminance value storage unit 1401. Remembered. Next, when the objective lens 5 is moved up by the movement pitch ΔZ by the Z moving mechanism 1409 and the objective lens 5 is moved to the height information Z (k + 1), all the pixels in the xy plane direction in the height information Z (k + 1) are obtained. Luminance values are acquired, and these luminance values I are stored in the luminance value storage unit 1401 in association with the height information Z (k + 1). Note that all the luminance values I acquired in one pixel after the objective lens 5 has moved all the moving distance are, for example, the relationship of I (luminance value) -Z (height information) in FIGS. 4A and 4B. Corresponds to each black circle on the curve of the graph.

  Then, the above-described operation is repeatedly performed within the preset moving distance, so that the maximum luminance value I and the maximum luminance value I corresponding to the maximum luminance value I are respectively obtained in all pixels in the xy plane direction. The information Z is obtained, and the information is combined to create an extended image or a three-dimensional image, which is displayed on the monitor 13 via the image transfer I / O 1410.

  As shown in FIG. 4A, when the luminance value I (k + 1) acquired this time is larger than the luminance value I (k) acquired last time, the luminance value I (k) stored in the luminance value storage unit 1401 and the high value The depth information Z (k) is updated to the luminance value I (k + 1) and the height information (k + 1). Conversely, as shown in FIG. 4B, the previously acquired luminance value I (k) is the luminance value I acquired this time. If it is larger than (k + 1), the luminance value I (k) and the height information Z (k) are stored in the luminance value storage unit 1401 as they are, and finally the luminance value stored in the luminance value storage unit 1401 An extended image or a three-dimensional image may be created using the value I and the height information Z.

  By the way, when an extended image or a three-dimensional image of the sample 6 having surfaces with different reflectivities as shown in FIG. 5 is created, the brightness adjustment value of the photodetector 9 is set for all movements of the objective lens 5 or the sample stage 6A. In common, as described above, there is a possibility that the brightness value I in a certain height information Z is insufficient or saturated and a part of the extended image is not created.

  Therefore, in the confocal laser microscope of the first embodiment, the luminance adjustment value calculation unit 1404 adjusts the luminance adjustment value for adjusting the detection sensitivity of the photodetector 9 every time the objective lens 5 moves, and the adjustment. The brightness adjustment value thus obtained is output to the detection sensitivity adjustment unit 10 via the brightness adjustment I / O 1405.

  For example, in the case where the value when the luminance value I is saturated is 100%, the adjustment processing of the luminance adjustment value is the maximum luminance value among the luminance values I corresponding to all the pixels in the xy plane direction. This is executed when I is not 80 to 60% (target luminance range). The target luminance range is variable.

  In the confocal laser microscope of the first embodiment, a photomultiplier is used as the photodetector 9 and the brightness adjustment value is set as the detection sensitivity G. In such a configuration, the detection sensitivity G can be adjusted by controlling the applied voltage HV applied from the detection sensitivity adjustment unit 10 to the photodetector 9, and is expressed as G = j × HV ^ a. Can do. Note that a is a constant (maker instruction value) representing the relationship between the detection sensitivity G and the applied voltage HV, and j is the number of dynodes (manufacturer instruction value), so a and j are photomultiplier solids, respectively. It becomes a value determined every time. Further, a may be obtained experimentally strictly for each apparatus.

For example, let us consider a case where a condition for setting the target level of the luminance value I to 80% of the dynamic range of the photodetector 9 is set as the control condition of the applied voltage HV.
When the dynamic range is 100%, the luminance value I (100%) is I (100%) = G (100%), assuming that the reflectance of the sample 6 is r and the optical system efficiency is t at the applied voltage HV (100%). ) × r × t, I (100%) = r × t × j × HV (100%) ^ a.

  If you want to adjust the brightness value I (80%), I (80%) = I (100%) x {HV (80%) ^ a / HV (100%) ^ a} Can be sought. Also, when a photodiode is used for the photodetector 9, the luminance adjustment value can be similarly obtained by calculation based on the characteristics of the amplifier. In general, since an amplifier is linear, when a photodiode is used for the photodetector 9, the calculation becomes easier.

  By the way, as described above, when a common luminance adjustment value is used within the moving distance of the objective lens 5 (FIG. 6A), the luminance value I (k + 1) among the total luminance values I in a certain pixel is maximized. However, if the brightness adjustment value is changed every time the objective lens 5 is moved under the same setting conditions (FIG. 6B), the brightness value I (k) among the total brightness values I in the same pixel is maximized. May end up. For this reason, when the brightness adjustment value is adjusted every time the objective lens 5 is moved, an extended image or a three-dimensional image may be generated based on the incorrect height information Z.

  Therefore, in the confocal laser microscope of the first embodiment, in order to obtain the correct height information Z even if the luminance adjustment value is adjusted every time the objective lens 5 is moved, the luminance value correlation calculation unit 1403 performs luminance adjustment. If it is determined that the brightness adjustment value has been adjusted by the value calculation unit 1404, the brightness value I before the brightness adjustment value is adjusted and the brightness value I after the brightness adjustment value are adjusted are correlated. Correction is performed on the luminance value I before the adjustment value is adjusted.

  For example, the applied voltage HV (k + 1) when the brightness value I (k + 1) is acquired at a certain height information Z (k + 1) and the brightness value I (k) is acquired at the previous height information Z (k). The applied voltage HV (k) is compared. At this time, if the deviation ΔHV between the applied voltage HV (k) and the applied voltage HV (k + 1) is not zero, the luminance value I (k) and the luminance value I (k + 1) are not correlated (luminance value). The luminance value correlation calculation unit 1403 determines that the luminance value before the luminance adjustment value is adjusted is determined to be that the luminance value I (k) and the luminance value I (k) are detected by different luminance adjustment values. Correction is performed by multiplying the value I by the ratio obtained from the context in which the luminance adjustment value has been adjusted. That is, using the above I (k) = r × t × j × HV (k) ^ a, the luminance value Itarget = {r × t × j × HV (k) ^ a} × {(HV (K + 1) ^ a) ÷ (HV (k) ^ a)} can be obtained.

  Thereby, as shown in FIG. 7, the luminance value I (k) acquired before the adjustment of the applied voltage HV and the luminance value I (k + 1) acquired after the adjustment of the applied voltage HV can be correlated. Therefore, even if the applied voltage HV is adjusted each time the objective lens 5 is moved, the correct height information Z can be obtained for all the pixels, and an accurate extended image or three-dimensional image can be created.

  FIG. 8 is a flowchart showing the operation of the controller 12 when all the luminance values I of the pixels in the xy plane direction are acquired at the movement distance. In addition, the said movement distance shall be preset.

  First, the luminance value I of each pixel in the xy plane direction at a certain height information Z is obtained from the photodetector 9 via the detection sensitivity adjustment unit 10 and is associated with the height information Z to obtain a luminance value storage unit. 1401 is stored (S101). The height information Z at the start of the operation of the controller 12 is, for example, the minimum value of the moving distance.

  Next, it is determined whether or not the luminance value I of each pixel stored in the luminance value storage unit 1401 is within the target luminance range (S102). Note that it may be determined whether some of the pixels stored in the luminance value storage unit 1401 are within the target luminance range.

If it is determined that it is not included (S102 is NO), the luminance adjustment value calculation unit 1404 adjusts the luminance adjustment value (applied voltage HV) (S103), and the process returns to S101.
On the other hand, if it is determined that it is present (YES in S102), the current luminance adjustment value is stored in the luminance adjustment value storage unit 1406 in association with the luminance value I acquired in S101 (S104).

Next, the current height information Z of the objective lens 5 is stored in the height information storage unit 1402 in association with the luminance value I acquired in S101 (S105).
Next, it is determined whether or not the deviation between the luminance adjustment value stored in the current luminance adjustment value storage unit 1406 and the luminance adjustment value stored in the previous luminance adjustment value storage unit 1406 is zero (S106).

  If it is determined that the deviation is not zero (NO in S106), the luminance value stored in the luminance value storage unit 1401 is acquired after adjustment of the luminance adjustment value for each luminance value I acquired before adjustment of the luminance adjustment value. Correction is performed so as to correlate with the luminance value I (S107).

  On the other hand, when it is determined that the deviation is zero (S106 is YES), it is determined whether or not the current height information Z is the upper limit value of the moving distance (S108). If it is determined that the value is not the upper limit (NO in S108), the objective lens 5 is moved up by the movement pitch ΔZ (S109), and the process returns to S101. Note that the sample stage 6A may be lowered by the movement pitch ΔZ.

  On the other hand, when it is determined that the value is the upper limit value (S108 is YES), the acquisition of the luminance value I is terminated. Then, the controller 12 obtains the largest luminance value I and the height information Z corresponding to the luminance value I from the luminance value storage unit 1401 and the height information storage unit 1402 in each pixel in the xy plane direction. These are extracted and combined to create an extended image or a three-dimensional image.

  According to the confocal laser microscope of the first embodiment, the brightness adjustment value is adjusted in real time during the creation of the extended image or the three-dimensional image, that is, every time the objective lens 5 or the sample stage 6A is moved. It is not necessary to adjust the brightness adjustment value by the user before creating the image or the three-dimensional image, and it is possible to suppress an increase in the time for creating the extended image or the three-dimensional image.

  Further, according to the confocal laser microscope of the first embodiment, since the brightness adjustment value is adjusted every time the objective lens 5 or the sample stage 6A is moved, all the brightness values I can be acquired correctly and are accurate. An extended image or a three-dimensional image can be created.

  Further, according to the confocal laser microscope of the first embodiment, the brightness adjustment value is adjusted every time the objective lens 5 or the sample stage 6A is moved, so that the brightness value I is set during the creation of the extended image or the three-dimensional image. There is no shortage or saturation, and there is no need to acquire the luminance value I from the beginning again.

  Moreover, according to the confocal laser microscope of 1st Embodiment, since it is not necessary to perform adjustment of a brightness | luminance adjustment value in advance, a user's burden can be eased. In the confocal laser microscope according to the first embodiment, the detection sensitivity G of the photodetector is controlled as a method for adjusting the brightness adjustment value, but the controller 12 adjusts the laser power as the method for adjusting the brightness adjustment value. The emission power of the laser light source 1 may be adjusted by controlling the drive current of the laser light source 1 via the DA converter 11 for use. In the case of such a configuration, it is assumed that the drive current of the laser light source 1 and the emission power have a linear relationship (1: 1).

  FIG. 9 is a flowchart showing the operation of the controller 12 in such a configuration. Of the operations of S201 to S209, operations other than S203 are the same as the operations of S101 to S109 except S103 shown in FIG.

  That is, when it is determined that the luminance value I of each pixel stored in the luminance value storage unit 1401 is not within the predetermined luminance range (NO in S202), the luminance adjustment value calculation unit 1404 determines the luminance adjustment value ( The output power of the laser light source 1 is adjusted (S203), and the process returns to S201.

  In the confocal laser microscope of the first embodiment, when the luminance value correlation calculation unit 1403 determines that the luminance adjustment value is adjusted by the luminance adjustment value calculation unit 1404, the luminance value I before the luminance adjustment value is adjusted. And the luminance value I before the luminance adjustment value is adjusted so that the luminance value I after the luminance adjustment value is adjusted is corrected. The luminance value I may be corrected. In this case, the luminance value Itarget to be corrected is obtained by Itarget = {r × t × j × HV (k + 1) ^ a} × {(HV (k) ^ a) ÷ (HV (k + 1) ^ a)}. Can do.

Second Embodiment
Next, a confocal laser microscope according to the second embodiment of the present invention will be described.
The confocal laser microscope of the second embodiment differs from the confocal laser microscope of the first embodiment shown in FIG.

  That is, in the confocal laser microscope according to the first embodiment shown in FIG. 1, it is determined whether or not the brightness adjustment value is adjusted every time the objective lens 5 or the sample stage 6A is moved. In the confocal laser microscope of the second embodiment, it is determined whether or not the brightness adjustment value has been adjusted after the objective lens 5 or the sample stage 6A has moved all the preset movement distances. When adjusted, the luminance value I is corrected.

  FIG. 10 is a flowchart showing the operation of the controller 12 in the confocal laser microscope according to the second embodiment. In addition, since each operation | movement of S301-S305 is the same as each operation | movement of S101-S105 shown in FIG. 8, description is abbreviate | omitted.

  It is determined whether or not the height information Z acquired this time is a preset upper limit value of the moving distance (S306). If it is determined that the value is not the upper limit (NO in S306), the objective lens 5 is moved up by the movement pitch ΔZ, and the process returns to S301.

  On the other hand, if it is determined that the value is the upper limit value (YES in S306), are all the brightness values I of each height information Z the same brightness adjustment value (detection sensitivity G or emission power of the laser light source 1)? It is determined whether or not (S307).

  If it is determined that they are not the same (NO in S307), correction is performed so that each luminance value I acquired before adjustment of the luminance adjustment value is correlated with each luminance value I acquired after adjustment of the luminance adjustment value. It performs (S308).

On the other hand, if it is determined that they are the same (YES in S307), the acquisition of the luminance value I is terminated.
Also in the confocal laser microscope according to the second embodiment, it is possible to suppress an increase in the time for creating an extended image or a three-dimensional image, similarly to the confocal laser microscope according to the first embodiment.

It is a figure which shows the confocal laser microscope of 1st Embodiment of this invention. It is a figure which shows a detection sensitivity adjustment unit. It is a figure which shows a luminance value correlation adjustment part. It is a graph which shows the relationship between a luminance value and height information. It is a graph which shows the relationship between a luminance value and height information. It is a figure which shows an example of a sample. It is a graph which shows the relationship between the luminance value when not adjusting a luminance adjustment value, and height information. It is a graph which shows the relationship between the luminance value and height information when adjusting a luminance adjustment value. It is a graph which shows the relationship between a luminance value and height information when correcting with respect to the luminance value acquired before adjustment of a luminance adjustment value. It is a flowchart which shows operation | movement of the confocal laser microscope of 1st Embodiment of this invention. It is a flowchart which shows operation | movement of the confocal laser scanning microscope of the modification of 1st Embodiment of this invention. It is a flowchart which shows operation | movement of the confocal laser microscope of 2nd Embodiment of this invention. It is a figure which shows the existing confocal laser microscope.

Explanation of symbols

1 Laser light source 2 PBS
3 Two-dimensional scanning mechanism 4 1 / 4λ plate 5 Objective lens 6 Sample 7 Condensing lens 8 Pin hole 9 Photo detector 10 Detection sensitivity adjustment unit 11 Laser power adjustment DA converter 12 Controller 13 Monitor 14 High-voltage power supply circuit 15 Amplifier 16 AD converter 17 DA converter 1401 Brightness value storage unit 1402 Height information storage unit 1403 Brightness value correlation calculation unit 1404 Brightness adjustment value calculation unit 1405 Brightness adjustment I / O
1406 Brightness adjustment value storage unit 1407 Z movement mechanism I / O
1408 Z moving mechanism driver 1409 Z moving mechanism 1410 Image transfer I / O

Claims (6)

A laser light source, an objective lens for condensing laser light emitted from the laser light source on the sample, a two-dimensional scanning means for two-dimensionally scanning the laser light on the sample, and the objective lens or the sample are mounted. And a moving means for moving the sample stage at regular intervals within a moving distance set in advance in the direction of the optical axis of the light emitted from the objective lens, and a position conjugate with the condensing position of the objective lens A confocal stop means and a photodetector for detecting the brightness of the light that has passed through the confocal stop means. Based on the brightness and the height information of the sample, an extended image or a three-dimensional image of the sample is obtained. A confocal laser microscope to create,
Adjusting means for adjusting at least one of the detection sensitivity of the photodetector or the emission power of the laser light source so that the luminance falls within a target luminance range;
When it is determined that at least one of the detection sensitivity or the output power is adjusted by the adjusting means while the objective lens or the sample stage is moving within the moving distance, the detection sensitivity or the output power Correlating means for correcting the luminance detected before or after adjustment to be correlated with the luminance detected before or after the detection sensitivity or the output power is adjusted;
A confocal laser microscope characterized by comprising: The confocal laser microscope according to claim 1,
The correlation means calculates the ratio of the luminance detected before the detection sensitivity or the output power is adjusted and the luminance detected after the detection sensitivity or the output power is adjusted to the detection sensitivity or the output power. A value obtained by multiplying the luminance detected before or after the adjustment is made as a value after correction of the luminance detected before or after the detection sensitivity or the output power is adjusted,
A confocal laser microscope characterized by that. The confocal laser microscope according to claim 1,
The correlation means determines whether at least one of the detection sensitivity or the output power has been adjusted for each movement of the objective lens or the sample stage at a certain interval.
A confocal laser microscope characterized by that. The confocal laser microscope according to claim 1,
The correlation means is configured to at least reduce the detection sensitivity or the output power while the objective lens or the sample stage is moving within the movement distance after the movement of the objective lens or the sample stage is completed. Determine whether one has been adjusted,
A confocal laser microscope characterized by that. The confocal laser microscope according to claim 1,
The photodetector is a photomultiplier or a photodiode;
The adjusting means adjusts the detection sensitivity by controlling a voltage applied to the photodetector;
A confocal laser microscope characterized by that. The confocal laser microscope according to claim 1,
The adjusting means adjusts the emission power by controlling a drive current to the laser light source;
A confocal laser microscope characterized by that.