JPH11271220A - Measurement parameter determining method for scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately determine measurement parameters by reducing the frequency of preliminary scanning, improving the measuring precision of a dynamic range and restricting the influence of noise. SOLUTION: An analog electric signal into which a reflected light, a fluorescent light or a transmitted light at the time when a laser light from a laser light source 1 is scanned to a specimen is photoelectrically transferred with a light receiving element 3 is converted into specimen image data via an offset computation part 4, an analog amplifier 5, and an A/D converter 6. Partial image data out of the specimen image data are extracted as samples by a sample extracting function 11 and the regular distribution of picture elements for brightness is derived from the sample groups by a regular distribution deriving function 12. The offset of the offset computation part 4 and the gain of the analog amplifier 5 are determined in accordance with a dynamic range of brightness obtained by the regular distribution by a measurement parameter determining function 13.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning microscope which converts a reflection image, a fluorescence image or a transmission image, which is a scan image of a specimen, into an electric signal by a light receiving element to form a sample image.
In particular, the present invention relates to a method for determining measurement parameters of a scanning microscope that optimizes the brightness of a sample image.

[0002]

2. Description of the Related Art In such a scanning microscope, first, a shutter is provided on an optical path leading to a light-receiving element for picking up a sample image, and an offset to an output of the light-receiving element is determined in a state where the shutter is closed to block light. ing.

[0003] Next, the shutter is opened, one sample image is picked up, the maximum luminance value and the minimum luminance value in the sample image are obtained from the output signal of the light receiving element, and the dynamic range (output current of the light receiving element) is wide. The gain and the ND filter value are increased by one step so that the capture of the image and the change of the measurement parameters are performed by comparing the difference between the maximum luminance value and the minimum luminance value of the image signal of the sample image with the maximum value of the input effective range of the scanning microscope. It repeats until it becomes.

However, in such a change of the measurement parameters, for example, when observing a sample image stained with a fluorescent dye, a gain is obtained for an image including light other than the sample, or a measurement of the gain or the like is performed. Since the sample is repeatedly scanned to set the parameters, the laser irradiation time may be long, and it may be buried in dark current noise.Therefore, a sufficient amount of light is not necessarily secured for the light receiving element. Did not.

[0005] Under such circumstances, for example, Japanese Patent Application No. 8-44.
No. 948 discloses a method for determining measurement parameters. In this method, as shown in FIG. 10, a laser beam emitted from a laser light source 1 is guided to a focusing optical system via a scanning optical system in a scanning microscope 2, and
Scan over the sample through the objective lens. The reflected light, fluorescence or transmitted light from the sample scanned by the laser light is received by the light receiving element 3 and converted into an analog electric signal and output. Then, the analog electric signal is adjusted in offset and gain by an offset calculator 4 and an analog amplifier 5, respectively, and then converted digitally by an A / D converter 6 and taken into a system control computer 7 as sample image data. The control computer 7 controls measurement parameters such as the offset of the offset calculation unit 4 and the gain of the analog amplifier 5.

In this method of determining measurement parameters, it is important to accurately measure a dynamic range (maximum value-minimum value) in order to determine a gain and an offset. Since the brightness is determined by the minimum value and the maximum value of each pixel, it is necessary to accurately measure the minimum value and the maximum value. The minimum and maximum values of these data are basically A /
Since the measurement cannot be made unless it is within the range that can be converted by the D converter 6, a preliminary scan is performed so as to fall within this range. This is because it is necessary to use the dynamic range of the A / D converter 6 to the maximum in order to measure the minimum value and the maximum value of data as accurately as possible. However, this scan is performed by trial and error in FIG.
The output current is controlled so that the maximum value of the data approaches the maximum value of the range of the A / D converter 6 by changing the control amount input to the light receiving element 3 shown in FIG.

[0007]

However, in the above-described method for determining the measurement parameters, since the accuracy of the dynamic range can only be (maximum value-minimum value), the light receiving element 3 is controlled and the maximum brightness is controlled. A / D converter 6
However, even if this dynamic range is set so as to be close to the dynamic range of the A / D converter 6 and measured under ideal conditions, a 12-bit A / D converter 6 is 4-digit, 16-bit A / D
Five digits are also significant digits in the converter.

Further, in order to accurately determine the minimum and maximum values of the brightness, a large amount of data must be extracted as samples from the sample image data constituting one screen and selected from them. The reason is that the probability that the maximum value and the minimum value are included in the partial data in the sample image data is low, and a preliminary scan must be performed in a wide range in order to increase the accuracy. This not only consumes time, but also causes fading, for example, when observing a fluorescent sample with a biological scanning microscope.

Further, the minimum value and the maximum value of data may be noise, and if the algorithm is continued as it is, an accurate dynamic range may not be obtained.
Therefore, the present invention provides a measurement parameter determination method for a scanning microscope that can reduce the number of preliminary scans, increase the measurement accuracy of the dynamic range, and suppress the influence of noise to determine an accurate measurement parameter. Aim.

[0010]

According to a first aspect of the present invention, a sample is scanned with a laser beam, light obtained from the sample is received by a light receiving element, and an analog electric signal output from the light receiving element is subjected to an offset calculating section. And converting it into digital sample image data through an analog amplifier, and determining the offset of the offset operation unit and the gain of the analog amplifier based on the sample image data. Extracting image data as a sample, deriving a normal distribution for the brightness of each pixel from a set of samples extracted in this step, and a dynamic range of brightness obtained from the normal distribution derived in this step. The offset of the offset calculation unit and the analog amplifier The measurement parameter determining method of a scanning microscope and a step of determining the in.

According to a second aspect of the present invention, in the method for determining a measurement parameter of a scanning microscope according to the first aspect, the number of samples extracted from the sample image data is standard deviation based on the brightness of the sample extracted from the sample image data. The standard deviation is used to determine the width of the confidence interval of the average using the image data of the sample image data as a population, and if the width of the confidence interval exceeds a preset reference value, the number of samples is determined. To increase.

According to a third aspect of the present invention, in the method for determining a measurement parameter of a scanning microscope according to the first aspect, when a plurality of normal distributions are created from the brightness of each pixel of the sample set, these normal distributions are determined. The thresholds to be separated are determined, the normal distributions are divided into groups, and the offset of the offset calculation unit and the gain of the analog amplifier are determined based on the dynamic range of brightness obtained using the respective normal distributions.

[0013]

(1) Hereinafter, a first embodiment of the present invention will be described with reference to the drawings. The same parts as those in FIG. 10 are denoted by the same reference numerals, and detailed description thereof will be omitted. FIG. 1 is a configuration diagram of a laser scanning microscope to which the measurement parameter determination method of the present invention is applied.

The system control computer 10 provides a command to the scanning microscope 2, the light receiving element 3, the offset operation unit 4, the analog amplifier 5, and the A / D converter 6 to set measurement parameters in each unit. And has a sampling function 11, a normal distribution deriving function 12, and a measurement parameter determining function 13.

The sample extracting function 11 has a function of extracting, as a sample, a part of image data of the sample image data taken in from the A / D converter 6. The normal distribution deriving function 12 has a function of deriving a normal distribution relating to the brightness of each pixel from a set of samples extracted by the sample extracting function 11.

The measurement parameter determination function 13 has a function of determining the offset of the offset calculation unit and the gain of the analog amplifier based on the dynamic range of brightness calculated using the normal distribution created by the normal distribution derivation function 12. Have.

Next, a method of determining each measurement parameter of the offset of the offset calculation unit 4 and the gain of the analog amplifier 5 in the laser scanning microscope configured as described above will be described.

The laser light emitted from the laser light source 1 is
The light is guided to a condensing optical system via a scanning optical system in the scanning microscope 2 and is scanned on a sample through an objective lens. Reflected light from the sample scanned by this laser light,
The fluorescent light or transmitted light is received by the light receiving element 3 and converted into an analog electric signal and output. The analog electric signal is adjusted in offset and gain by the offset operation unit 4 and the analog amplifier 5, respectively, and is then digitally converted by the A / D converter 6 and taken into the system control computer 7 as sample image data. .

First, the system control computer 7 controls the amount of input to the light receiving element 3, and here is fixed at a value determined to be optimal in advance. Next, the dynamic range of the output current from the light receiving element 3 is determined using the sample image data. That is, the sample extraction function 11 extracts a part of the image data of the sample image data as a sample. Each pixel data (pixel data) constituting the target sample image data indicates the brightness of the sample to be viewed, and all the pixel data are considered as a population.

There are various methods for extracting a sample from a population consisting of all pixel data. For example, as shown in FIG. 2, scanning is performed on the diagonal line of the sample image data 14 (preliminary scan) to obtain the pixel data. Collect Alternatively, as shown in FIG. 3, for example, approximately three horizontal line portions (rasters) of the sample image data 14 are selected to collect pixel data,
As shown in FIG. 4, there is a method of extracting each pixel data at a completely random place. Some systems allow the end-user to choose how to take the specimen. However, a preliminary scan is performed according to some rules, and brightness data at a necessary position is adopted as a sample.

The precision of the A / D converter 6 is 16 bits. As a result, the brightness data becomes 0-65535.
, And data that does not fall within this brightness range, that is, data that is too dark and data that is too bright are each 0,65535.

Next, a normal distribution for the brightness of each pixel is derived from the set of samples extracted by the normal distribution deriving function 12. That is, an average value x and a standard deviation s are obtained to derive a normal distribution from the extracted sample. However,
Data with a brightness of “0” is excluded from the sample. This is because the data is likely not from the sample but from the background. In the present embodiment, only data having a brightness of “0” is excluded. However, the present invention is not limited to this, and a certain range such as excluding brightness data of “0 to 10” may be excluded.

Next, if the average value x is at least half of the maximum value (hereinafter, referred to as AD _max ) that can be represented by the A / D converter 6, that is, if x> AD _max / 2 (1), the light receiving element 3 is turned off. Control so that its output current decreases. In the present embodiment, since the gain range of the analog amplifier 5 is 1 to 10, 1/10 times, 1/1
The light receiving element 3 may be controlled in the directions of 00 times and 1/1000 times until x <AD _max / 2 (2) is satisfied.

As a result, a normal distribution as shown in FIG. 5 is derived. Next, the offset of the offset calculator 4 and the gain of the analog amplifier 5 are determined by the measurement parameter determination function 13 based on the dynamic range of brightness obtained from the normal distribution. That is, first, it is determined how much of the sample needs to be captured as valid data. The valid data means that the brightness does not collapse to “0” and does not exceed AD _max . In this embodiment, assuming that 99.7% of data is required as valid data, the valid data is x ± 3s from the normal distribution.

(3) Therefore, when the maximum value of the brightness of the sample _{I max,} the minimum value and I _min, the _{I max = x + 3s ... (} 4) I min = x-3s ... (5). Here, (I _max -I _min ) is defined as a dynamic range.

If the dynamic range is smaller than a predetermined value, the light receiving element 3 is controlled to amplify the output current. For example, dynamic range, determines that it is necessary to control the _{(I max -I min) <100} ... (6) If light-receiving element 3. In that case, the output current of the light receiving element 3 is 10 times, 100 times,
The light receiving element 3 is controlled in the direction of 1000 times until (I _max -I _min )> 100 (7) holds.

Next, a new offset o and gain g are obtained based on the dynamic range. These offsets o, the gain g computes _{g = (AD max / (I} max -I min)) * g2 / k ... (8) o = ((I min + o2) / g2) * g ... (9) It is required by Here, o2 is the original offset and g2 is the gain.

In the present embodiment, since 1 <g <10 must be satisfied, if the value does not fall within this range,
The output current is controlled using the light receiving element 3. That is, the light receiving element 3 is controlled so that the output current of the light receiving element 3 changes in units of 10 times, and the magnification of the remaining output current is controlled by the gain g of the analog amplifier 5. Also, k can be normally "1", but is used when it is desired to suppress the output result to a certain bit width. For example, when it is desired to reduce the number to 8 bits, k = 2 ＾ ｛(A
/ D converter 6 bit width)-(required data bit width)｝
= 2 ＾ (16−8).

For example, if the light receiving element 3 is a photomultiplier, the effective current amplification is 10K to 100M times, so the width is 10K, which is within the dynamic range of the 16-bit A / D converter 6. . (K = 10 ³ ,
M = 10 ⁶ ) Therefore, since the average value is usually x> AD _max / 2, the output current of the light receiving element 3 is reduced or the dynamic range (I
_max -I _min ) is too small to amplify the output current of the light receiving element 3.

As described above, in the first embodiment, a part of the sample image data is extracted as a sample, and a normal distribution for the brightness of each pixel is derived from the extracted sample set. since determining the offset and gain of the analog amplifier 5 of offset calculating section 4 based on the dynamic range of brightness obtained from the normal distribution, to determine the dynamic range (I _max -I _min) in a single preliminary scan , Light receiving element 3, offset and gain measurement parameters can be set. The user does not need to set anything while setting these measurement parameters, and 95.4% of the data is too dark (= 0),
It can be used effectively without being too bright (= 65535). Further, since the range of the preliminary scan is small for all the sample image data, the user can see the result quickly, and the discoloration can be suppressed accordingly. (2) Hereinafter, a second embodiment of the present invention will be described with reference to the drawings. The configuration of the laser scanning microscope according to the second embodiment is the same as that of the laser scanning microscope shown in FIG.

The sample extracting function 11 in the laser scanning microscope has a function of determining whether the number of samples is appropriate and increasing the number of samples if the number of samples does not reach the standard.

Next, the operation of determining the number of samples in the laser scanning microscope configured as described above will be described with reference to the sample extraction flowchart shown in FIG. First, the control of the input amount to the light receiving element 3 is fixed to a value determined in advance to be optimum here.

Next, the dynamic range of the output current from the light receiving element 3 is determined using the sample image data. Each pixel data constituting the target sample image data indicates the brightness of the sample to be viewed in the same manner as described above, and all the pixel data are considered as a population.

The method of extracting a sample from the population set including all pixel data is performed by the sample extracting function 11 in step #.
1, sample image data 1 as shown in FIG.
For example, a method of selecting four rasters at about three places and collecting the pixel data is used.

Therefore, as shown in FIG. 7, for example, three points "1" to "3" are selected as rasters in the sample image data 14, and their pixel data are collected, and an average value x is obtained using these pixel data.

Then, in step # 2, the width of the 95.4% confidence interval of the average value of the population is calculated using the t distribution. That is, a 95.4% confidence interval x ± a of the average value of the population is obtained.

Assuming that the number of samples obtained from the three rasters "1-3" is n, the standard error is e, and the standard deviation of the samples is s, the standard error e is e = s / (n) ^{1 / 2} … (10)

The t value of the confidence interval 95.4% is 4.303
Thus, a = (t value) * e (11)

Next, the validity of the number of samples is evaluated by this a. In step # 3, a is compared with a preset reference value, and if a exceeds this reference value,
Scanning of the sample image data 14 is performed by increasing the number of rasters on the sample image data 14 shown in FIG.

When the scan is performed at the raster locations "4 to 6", the sample extracted at the raster locations "1 to 6" combined with the first scans "1 to 3" at step # 2 is re-reliable. The section a is obtained and compared with the reference value in step # 3. If a satisfies the standard, it is determined that the number of samples is sufficient.

In the following, similarly to the first embodiment, the offset of the offset calculator 4 and the gain of the analog amplifier 5 are determined based on the dynamic range of brightness obtained from the normal distribution.

In the sample extraction flowchart shown in FIG. 6, the number of loops is not determined. However, the number of loops may be repeated forever until the standard is reached, or the number of loops may be determined, for example, five. Also, for example, even if the loop is performed five times, a
If does not reach the reference value, it may be considered that the histogram of the sample cannot be approximated by a normal distribution, and the measurement parameters may be automatically set by a conventional method.

As described above, in the second embodiment, it is needless to say that the same effects as those of the first embodiment can be obtained, and whether the number of samples is appropriate is determined. If has not reached the standard, it has a function of increasing the number of samples, so that the number of samples can be dynamically changed. In other words, if it is determined that the number of samples per sample is sufficient to obtain the dynamic range, the sample image data can be quickly acquired. Dynamic scanning can be performed and dynamic range accuracy is guaranteed. (3) Hereinafter, a third embodiment of the present invention will be described with reference to the drawings. Note that the configuration of the laser scanning microscope of the third embodiment is the same as that of the laser scanning microscope shown in FIG. 1, and therefore, will be described with reference to FIG.

When there are a plurality of normal distributions created from the brightness of each pixel of the sample set, the measurement parameter determination function 13 in the laser scanning microscope determines each threshold value for dividing the normal distributions. Each normal distribution is divided into groups, and the function of determining the offset of the offset calculation unit 4 and the gain of the analog amplifier 5 based on the average value and the standard deviation for each normal distribution is provided.

Next, the operation when a plurality of normal distributions exist in the laser scanning microscope configured as described above will be described. First, the control of the input amount to the light receiving element 3 is fixed to a value determined in advance to be optimum here.

Next, the dynamic range of the output current from the light receiving element 3 is determined using the sample image data. Each pixel data constituting the target sample image data indicates the brightness of the sample to be viewed in the same manner as described above, and all the pixel data are considered as a population.

In the method of extracting a sample from the population comprising all pixel data, the sample extracting function 11 scans the sample image data 14 on a diagonal line as shown in FIG.

Then, an average value x and a standard deviation s are obtained from these extracted samples, and the normal distribution thereof is derived. For example, when observing a sample of a metal piece with reflected light with an industrial scanning microscope, the brightness of the reflected light may be determined by various angles of the metal piece. For example, if the surface of the metal piece has an angle of 30, 45, or 60 degrees from the horizontal plane, the histogram obtained from the extracted sample has a distribution as shown in FIG.

Since this histogram has three brightness groups, each of the threshold values f ₁ ,
determine the f _2. These threshold values f ₁ and f ₂ may be manually input by the user in advance, or a histogram of the brightness distribution may be created, and the threshold values f ₁ and f ₂ may be calculated from discrete portions in the histogram.
₁ and f ₂ may be obtained. That is, the threshold values shown in FIG. 8 are threshold values f ₁ and f ₂ determined by some method.

Here, the normal distribution is derived in three groups. With these normal distributions Q ₁ , Q ₂ , Q ₃ , their average values and standard deviations are x ₁ , x ₂ , x
₃ , s ₁ , s ₂ , and s ₃ .

Since the end user usually wants to see all brightness data, it is assumed that I _max = x ₃ + 3 · s ₃ (12) I _min = x ₁ −3 · s ₁ (13)

When it is desired to limit only to the normal distributions Q ₂ and Q ₃ , I _max = x ₃ + 3 · s ₃ (14) I _min = x ₂ −3 · s ₂ (15)

Thus, from each normal distribution, I _max , I _{min
Are determined} , the respective offsets of the offset calculator 4 and the respective gains of the analog amplifier 5 are determined based on these I _max and I _min .

As described above, in the third embodiment, it goes without saying that the same effects as those in the first embodiment can be obtained, and each threshold for dividing the normal distributions Q ₁ , Q ₂ , and Q _3. The values f ₁ and f ₂ are determined, and these normal distributions Q
Since determining the offset and gain of the analog amplifier 5 of offset calculating section 4 based on the _1, Q _2, Q _3, even when the normal distribution Q _1, Q _2, Q ₃ there are a plurality of these normal distribution Q _1, By using the mean and standard deviation of each of Q ₂ and Q ₃ in combination, the offset,
Automatic setting of gain measurement parameters.

The present invention is not limited to the first to third embodiments, but may be modified as follows. For example, a sample is extracted in the first embodiment, it is determined whether the sample is distorted with respect to the normal distribution, and the maximum value I _max and the minimum value I _min are determined according to the amount of the distortion.
May be modified to change and set the dynamic range.

Hereinafter, this method will be described. In addition,
Such a laser scanning microscope has the same configuration as that of the laser scanning microscope shown in FIG. 1 and will be described with reference to FIG.
First, the control of the input amount to the light receiving element 3 is fixed to a value determined in advance to be optimum here.

Next, the dynamic range of the output current from the light receiving element 3 is determined using the sample image data. Each pixel data constituting the target sample image data indicates the brightness of the sample to be viewed in the same manner as described above, and all the pixel data are considered as a population.

The method of extracting a sample from a population consisting of all pixel data is performed by, for example, scanning the sample image data 14 on a diagonal line as shown in FIG. For example, as shown in FIG. 3, the raster of the sample image data 14 is selected at, for example, about three places to collect its pixel data, or as shown in FIG. 4, each pixel data at a completely random place is extracted. It may be a method.

Assuming that the number of samples thus extracted is n, an average value x and a standard deviation s are obtained, and a skewness w is obtained from the average value x and the standard deviation s. This skewness w is Is represented by Here, I _i is each sample (brightness) data.

As a result, the skewness w represents a deviation from the normal distribution as shown in FIGS. 9A and 9B. FIG. 9A shows the case where w> 0, and FIG. <0. Next, in each of the above embodiments, the maximum value of the brightness in the normal distribution is I _max , and the minimum value is I _min , and I _max = x + z I _min = x−z. _max , from the minimum value I _min to (I
_max -I _min ).

On the other hand, in this modified example, the maximum value of the brightness in the normal distribution is I _max , the minimum value is I _min , and I _max = x + z ₂ (17) I _min = x−z ₁ ( 18) and a dynamic range (I _max -I _min ) from these I _max and I _min .

Here, z ₂ and z ₁ are defined as follows. z ₂ = z + m * w (19) z ₁ = z−m * w (20) m is a predetermined positive constant, which enables adjustment of the displacement amounts of z ₂ and z ₁ . Then, in the case of w> 0 shown in FIG. 9A, z ₁ <z ₂ , and in the case of w <0 shown in FIG. 9B, z ₁ > z ₂ , and the population deviates from the normal distribution. The dynamic range can be determined in consideration of the above.

In a sample in which the tendency of distortion is known in advance,
The value of m may be determined for each type, and the user may manually input the value of m. Based on the maximum value I _max and the minimum value I _min thus determined, each offset of the offset calculation unit 4 and each gain of the analog amplifier 5 are determined in the same manner as in the first embodiment.

As described above, it is determined whether the sample is distorted with respect to the normal distribution, and the maximum value I is determined according to the amount of the distortion.
_{Since the maximum} value and the minimum value _Imin are corrected, it is possible to set the measurement parameters of the sample image data having a distortion with respect to the normal distribution. As a result, the maximum value of the brightness I
_{One of max} and the minimum value I _min exceeds the range of valid data, and the other is set in the valid data, thereby preventing excess or deficiency of valid data of the population. That is,
When sample image data is acquired with new measurement parameters, it is possible to prevent measurement of an image having a lot of dark and invalid data or an image in which a bright portion is skipped.

[0064]

As described above in detail, according to the first to third aspects of the present invention, the number of preliminary scans is reduced, the measurement accuracy of the dynamic range is increased, and the influence of noise is suppressed to achieve accurate measurement. A method for determining measurement parameters of a scanning microscope capable of determining parameters can be provided.

According to the second aspect of the present invention, the preliminary scanning is repeatedly performed until a satisfactory result for obtaining the dynamic range is obtained, and the measurement parameters of the scanning microscope capable of guaranteeing the accuracy of the dynamic range are obtained. A decision method can be provided.

Further, according to the third aspect of the present invention, it is possible to provide a method for determining measurement parameters of a scanning microscope which can automatically set offset and gain measurement parameters even when a plurality of normal distributions exist.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing a first embodiment of a laser scanning microscope to which a measurement parameter determination method according to the present invention is applied.

FIG. 2 is a schematic diagram showing a method of extracting pixel data by scanning on a diagonal line of sample image data.

FIG. 3 is a schematic diagram showing a method of selecting pixel data from three or so rasters of sample image data and extracting pixel data;

FIG. 4 is a schematic diagram showing a method for extracting pixel data of a completely random portion of sample image data.

FIG. 5 is a diagram showing a normal distribution of brightness data.

FIG. 6 is a sample extraction flowchart of the laser scanning microscope according to the second embodiment of the present invention.

FIG. 7 is a schematic diagram showing a sample extracting operation of the laser scanning microscope.

FIG. 8 is a diagram showing a plurality of normal distributions observed by a laser scanning microscope according to a third embodiment of the present invention.

FIG. 9 is a diagram showing the skewness of a normal distribution.

FIG. 10 is a view for explaining a method for determining measurement parameters of a conventional scanning microscope.

[Explanation of symbols]

 1: laser light source, 2: scanning microscope, 3: light receiving element, 4: offset operation unit, 5: analog amplifier, 6: A / D converter, 10: computer for system control, 11: sampling function, 12: Normal distribution derivation function, 13: Measurement parameter determination function.

Claims (3)

[Claims] 1. A sample is scanned by a laser beam, light obtained from the sample is received by a light receiving element, and an analog electric signal output from the light receiving element is converted into digital sample image data through an offset operation unit and an analog amplifier. And a method for determining a measurement parameter of a scanning microscope that determines an offset of the offset calculation unit and a gain of an analog amplifier based on the sample image data, wherein a part of the sample image data is extracted as a sample. Deriving a normal distribution for the brightness of each pixel from the set of samples extracted in this step; and the offset calculating unit based on the dynamic range of the brightness obtained from the normal distribution derived in this step. Offset and analog amplifier gain Determining a measurement parameter of the scanning microscope. 2. The sample number extracted from the sample image data, a standard deviation is obtained based on the brightness of the sample extracted from the sample image data, and the image of the sample image data is calculated using the standard deviation. 2. The scanning method according to claim 1, wherein a width of a confidence interval of an average value obtained by using data as a population is obtained, and if the width of the confidence interval exceeds a predetermined reference value, the number of samples is increased. Method for determining measurement parameters of scanning microscope. 3. When there are a plurality of normal distributions created from the brightness of each pixel of the sample set, each threshold value for dividing the normal distributions is determined, and each normal distribution is divided into each group. 2. The measurement parameter of the scanning microscope according to claim 1, wherein the offset of the offset calculation unit and the gain of the analog amplifier are determined based on the dynamic range of the brightness obtained using the respective normal distributions. Decision method.