JPH09236750A - Determining method measuring parameter of scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To minimize the number of times of scanning required for determining a parameter and to reduce the damage of a sample. SOLUTION: This method is the determining method of a measuring parameter of a scanning microscope and includes the following step. An input control amount capable of setting the output of a photodetector 3 is found in plural stages, and the image data of a sample are fetched by setting the input control amount obtained from the photodetector 3. Changing of the input control amount is repeated until the maximum value of the image data of the sample satisfys a prescribed condition and the input control amount suitable for finding the dynamic range is selected. From the maximum value convertible to a digital value by an A/D converting means 6 and the dynamic range obtained, an offset and a gain are determined.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning microscope in which a fluorescence image, a transmission image, or the like, which is a scanned image of a sample, is converted into an electric signal by a light receiving element to form a sample image. It relates to a method for determining measurement parameters to optimize.

[0002]

2. Description of the Related Art Currently, scanning microscopes include a biological laser scanning microscope used for observing biological specimens and an industrial laser scanning microscope used for inspection of semiconductor wafers. In order to maximize the ability of any scanning microscope, various measurement parameters must be optimally adjusted according to the condition of the sample.

Conventionally, in an industrial laser scanning microscope, a shutter is provided on an optical path leading to a light receiving element for picking up a sample image, and an offset with respect to an output of the light receiving element is determined in a state where the shutter is closed and light is blocked. . Next, the shutter is opened, one sample image is captured, 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 gain and the filter value of the ND filter are set so that the dynamic range becomes wide. This step is repeated by one step, and this image capture and measurement parameter change are repeated until the difference between the maximum luminance value and the minimum luminance value of the image signal of the sample image reaches the maximum effective input range of the scanning microscope. Also in the biological scanning microscope, the measurement parameters are set in the same manner as in the above-mentioned industrial laser scanning microscope.

[0004]

However, when the measurement parameters of the biological scanning microscope are determined by the same procedure as the industrial laser scanning microscope, the following problems occur. First, when observing a specimen image in which the specimen is stained with a fluorescent dye, the solution immersed in the specimen or the cover glass holding the specimen emits light. The user needs to change the relative brightness by subtracting the brightness of non-cells when actually observed, rather than the offset that eliminates electrical noise. I was looking for auto gain.

Secondly, when observing a biological specimen with fluorescence,
As the laser irradiation time increases, the damage to the sample increases, so it is desirable to reduce the irradiation time, that is, the number of measurements as much as possible. However, the laser irradiation time is long because the sample is repeatedly scanned to set measurement parameters such as gain.

Thirdly, since biological specimens are generally darker than metal specimens, unless the amount of light incident on the light-receiving element is secured, the image signal may cause dark current noise due to the light-receiving element. Although there is a possibility that the light will be buried, it has not always been possible to secure a sufficient amount of light for the light-receiving element because the parameters have been manually determined by trial and error in the past.

The present invention has been made in view of the above situation, and minimizes the number of scans required to determine a measurement parameter to reduce the damage to the sample and facilitates the parameter setting operation. Can reduce the burden on the user,
It is an object of the present invention to provide a method for determining a measurement parameter that can surely determine an optimum measurement parameter.

[0008]

In order to achieve the above object, the present invention takes the following measures. The present invention corresponding to claim 1 guides a laser beam emitted from a laser light source to a focusing optical system via a scanning optical system to scan a sample,
After the reflected light or the transmitted light from the sample scanned with the laser light is incident on the light receiving element and converted into an analog electric signal, and the analog electric signal is adjusted in the offset adjustment unit and the analog amplification unit, respectively, In a scanning microscope in which a signal converted into a digital signal by the D / D conversion means is taken in as sample image data, and measurement parameters such as light receiving sensitivity, offset and gain of the light receiving element, the offset adjusting section and the analog amplifying section are controlled from a control computer. Is a method of determining measurement parameters,
(A) a step of obtaining an input control amount capable of setting the output of the light receiving element in a plurality of stages, (b) a step of setting the input control amount obtained in step (a) to the light receiving element and loading sample image data, c) changing the input control amount until the maximum value of the sample image data satisfies a predetermined condition and repeating step (b) to select an input control amount suitable for obtaining the dynamic range of the output current of the light receiving element, (D) a step of setting the selected input control amount in the light receiving element and measuring the dynamic range of the output current, (e) a maximum value that can be digitally converted by the A / D conversion means, and the step (d) Determining the offset and gain from the dynamic range.

According to the present invention, the number of scans of the sample is only a predetermined number for determining the input control amount set in the light receiving element, and the number of scans for determining the gain and the offset can be reduced. The damage done can be reduced. Further, since the sensitivity is set for the light receiving element in accordance with the effective input range of the A / D conversion means and other parameters are determined, it is possible to prevent the analog light receiving signal from being buried in the dark current of the light receiving element. .

According to a second aspect of the present invention, the laser light emitted from the laser light source is guided to a condensing optical system via a scanning optical system to scan a sample, and the sample is reflected by the laser beam. Light or transmitted light is incident on the photomultiplier and converted into an analog electric signal, and the analog electric signal is adjusted with an offset adjustment unit and an analog amplification unit, respectively, and then converted into a digital signal with A / D conversion means. Captured signal as sample image data, the photomultiplier from the control computer, the applied voltage of the offset adjustment unit and the analog amplification unit, a method of determining the measurement parameters in the scanning microscope to control the measurement parameters such as offset and gain,
(A) a step of obtaining an applied voltage capable of setting the current multiplication factor of the photomultiplier in a plurality of stages, (b) a step of setting the applied voltage obtained in step (a) to the photomultiplier and capturing sample image data , (C)
Changing the applied voltage until the maximum value of the sample image data satisfies a predetermined condition and repeating step (b) to select an applied voltage suitable for obtaining the dynamic range of the output current of the photomultiplier, (d) Setting the selected applied voltage to the photomultiplier and measuring the dynamic range of the output current, (e)
The method further comprises the step of determining the offset and gain from the maximum value that can be digitally converted by the A / D conversion means and the dynamic range obtained in step (d).

According to the present invention, after the applied voltage suitable for measuring the dynamic range of the output current of the photomultiplier is determined, the dynamic range of the output current is measured with the applied voltage fixed. Offset and gain can be calculated by using this dynamic range.

According to a third aspect of the present invention, a laser beam emitted from a laser light source is guided to a focusing optical system through a scanning optical system to scan a sample, and the sample is reflected by the laser beam. Light or transmitted light is incident on the light receiving element to be converted into an analog electric signal, and the analog electric signal is adjusted in offset and gain by an offset adjusting section and an analog amplifying section, respectively, and then converted into a digital signal by A / D converting means. A method of determining a measurement parameter in a scanning microscope for controlling a measurement parameter such as a light receiving element, an input light of an offset adjusting unit and an analog amplifying unit, a measurement parameter such as an offset and a gain from a control computer by capturing a signal as sample image data. ) A step of obtaining an input light intensity capable of setting the output of the light receiving element in a plurality of stages, (b) The step of capturing sample image data by entering the amount of light the input light intensity obtained in (a),
(C) changing the input light intensity until the maximum value of the sample image data satisfies a predetermined condition, and repeating step (b) to select an input light intensity suitable for obtaining the dynamic range of the output current of the light receiving element. , (D) setting the selected input light intensity in the light receiving element and measuring the dynamic range of the output current, (e) the maximum value that can be digitally converted by the A / D conversion means, and step (d)
And a step of determining the offset and the gain from the dynamic range obtained in step 1.

According to the present invention, the amount of light incident on the light receiving element is determined to have an intensity suitable for measuring the dynamic range of the output current of the light receiving element, and then fixed to the input light intensity to fix the dynamic range of the output current. Is measured. Offset and gain can be calculated by using this dynamic range.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. (First Embodiment) FIG. 1 shows the system configuration of a laser scanning microscope. In this laser scanning microscope, a laser beam generated by a light source 1 composed of a laser device is used for scanning laser microscope 2
The laser light which is incident on the scanning optical system and passes through the scanning optical system is scanned in a predetermined direction, and then the sample is irradiated through the objective lens. After the reflected light or the transmitted light from the sample is converted into an electric signal by the light receiving element 3, the analog electric signal is input to the offset calculation unit 4 to add the set offset. Further, after the analog electric signal output from the offset calculation unit 4 is input to the analog amplifier 5 and adjusted by the set gain, the analog signal is converted into a digital signal by the A / D converter 6 and then to the system control computer 7. send. The system control computer 7 displays a sample image obtained by processing the digital signal, and performs necessary analysis processing.

The system control computer 7 sets a measurement parameter in each part by giving a command to the scanning microscope 2, the light receiving element 3, the offset calculation part 4, the analog amplifier 5, and the A / D converter 6. . The output current of the light receiving element 3 is controlled by the input control amount for sensitivity adjustment given from the system controlling computer 7. Further, the offset calculator 4 changes the offset amount to be added / subtracted to / from the input analog signal according to the offset setting command given from the system control computer 7. Further, the analog amplifier 5 changes the amplification factor for the input analog signal according to the gain setting command given from the system control computer 7.

Next, a method of determining each measurement parameter of the light receiving sensitivity (PS) of the light receiving element 3, the offset amount of the offset calculator 4, and the gain amount of the analog amplifier 5 in the scanning microscope configured as described above. explain.

First, the range of the light receiving sensitivity of the light receiving element 3 within which the output current of the light receiving element 3 is within the effective range at the input to the A / D converter 6 is obtained. An input control amount that can set the output current of the light receiving element 3 in a plurality of stages that can be linearly controlled within the range of the light receiving sensitivity is selected.

FIG. 2 shows the relationship between the input control amount and the output current for determining the light receiving sensitivity of the light receiving element 3. Light receiving element 3
, The output current is increased by increasing the light receiving sensitivity, but the change is non-linear. The light receiving element 3 is
It can be composed of a PMT (photomultiplier) described later, a photodiode, or the like.

The characteristic graph shown in FIG. 2 is measured for the light receiving element 3. The output current value taken on the vertical axis on this characteristic graph is equally divided into N in an arbitrary range. In the figure, the non-linear output of the light receiving element is divided into N equal parts so as to be linearly controllable by an integral multiple. Each N-divided position becomes a position of a plurality of stages of the output current, such as double, triple, ... In order based on the lowest current value. Then, each N-divided position on the graph is reversely pulled to the horizontal axis side, and each corresponding horizontal axis position becomes the value of the input control amount with which the output current can be set by an integral multiple. In this specification, such a value of the input control amount is referred to as "PS rank". In the example shown in FIG. 2, the PS ranks of 1 to 10 are set so that the output current can be multiplied by an integer between 1 and 10 times. Specifically, the output current when PS rank = 2 is twice the output current when PS rank = 1, and the output current when PS rank = 3 is triple the output current when PS rank = 1. When this magnification is n times, the electrical gain is 1 to m (n ≦ n
m) Up to 2 times can be set. As a result, the gain can cover the discrete range between the ranks of the light receiving sensitivity.

Next, the dynamic range of the output current from the light receiving element 3 is obtained using the sample image. Determine the PS rank for measuring this dynamic range.
It is necessary to prevent the value after A / D conversion including the maximum brightness of the captured image from overflowing. for that reason,
The light receiving sensitivity of the light receiving element 3 is such that the maximum value I _max of the output current corresponding to the maximum brightness in the sample image is as close as possible to the maximum value AD _max that can be digitally converted by the A / D converter 6 and does not exceed AD _max. Must be set to.

FIG. 3 is a flow chart for obtaining the PS rank for measuring the dynamic range of the output current of the light receiving element 3. In the process of step S1, initial values of measurement parameters (PS rank, gain, offset) are set. PS rank = 5 is set
This is to perform the work starting point from among the PS ranks 1 to 10 selected in the above work. Initial values of the measurement parameters are set in the respective parts by the system control computer 7.

In the process of step S2, the entire sample is scanned only once to capture the sample image. The system control computer 7 gives a scan command to the scanning microscope 2 to scan the sample two-dimensionally. An analog signal corresponding to the sample image obtained by the two-dimensional scanning is passed through each unit in which the various parameters set in step S1 are set, and then is fetched from the A / D converter 6 into the system control computer 7.

In the process of step S3, step S
The maximum value I from the image data of the sample image acquired in 2
_{Determine max} and minimum value I _min . Then, step S4
In seeking the number of I _max exceeding AD _max, further the number of I _max exceeding AD _{max (N-over-)} a predetermined value (N
_limit ) is judged.

If the condition of N _over > N _limit is satisfied, it is determined by the process of step S5 whether or not the light receiving sensitivity can be made higher than that at present. In the above setting, the maximum value of the light receiving sensitivity is PS rank = 10, so PS rank <
If the condition of 10 is satisfied, the light receiving element 3 is instructed to increase the rank of the input control amount (step S6). After that, the sample is two-dimensionally scanned again, and the light is received by the light receiving element 3 whose PS rank is increased by one rank. The output of the light receiving element 3 at this time is fetched from the A / D converter 6 to the system control computer 7. Then, the maximum value I _max and the minimum value I _min are calculated again from the image data of the sample image acquired in step S7 (step S8), and the number of I _max exceeding AD _max is calculated (step S9). As a result, N _over > N _limit
If the above condition is satisfied, the PS rank one time before is finally selected as the light receiving sensitivity to be set in the light receiving element 3.

On the other hand, in the processing of step S4, N
_{When over} > N _limit is not established, step S11
Then, it is determined whether or not the PS rank can be further lowered by shifting to the processing of. Since the PS rank can be lowered to PS rank = 1, the input control amount is lowered by one rank while PS rank> 1 holds. After that, the sample is two-dimensionally scanned again, and is received by the light receiving element 3 whose input control amount is lowered by one rank. The output of the light receiving element 3 at this time is fetched from the A / D converter 6 to the system control computer 7. Then, again, the maximum value I _max and the minimum value I _min are obtained from the image data of the sample image acquired in step S13 (step S1
4), the number of I _max exceeding AD _max is N _over ≦ N
_{When the limit} is reached, the light receiving sensitivity corresponding to the PS rank at that time is selected (step S15).

[0026] By the above processing, the maximum value I _max is the closest, and the light receiving sensitivity of the controllable light-receiving element 3 so as not to exceed the AD _max in AD _max of the output current of the light receiving element 3 (PS
Rank) has been decided.

Therefore, P determined as described above
An input control amount corresponding to the S rank is set in the light receiving element 3, the sample is scanned, and the dynamic range of the output current of the light receiving element 3 at that time is measured.

Next, the gain value of the analog amplifier 5 and the offset value of the offset calculator 4 are determined. In order to make the most efficient use of the functions of the A / D converter 6 for the most efficient conversion, I _max should be close to AD _max and I _min should be 0.
It is better to set parameters that approach This means that the gain should be set so that the dynamic range of the current signal matches the cover range of the A / D converter 6. If the A / D converter is 12 bits, I _max will be close to AD _max = 4095 and I _min will be close to 0.

FIG. 4 shows a flowchart for determining the gain value and the offset value. In the process of step T1, the gain V _gain of the analog amplifier 5 is calculated from the maximum value AD _max of the A / D converter 6 and the dynamic range (D) of the output current of the light receiving element 3. Here, the dynamic range (D) of the output current is
It is the dynamic range of the output current output from the light receiving element 3 when the sample image is taken with the light receiving sensitivity selected by the above method.

In the processing of step T2, the gain V _gain obtained in step T1 is compared with the maximum value G _max of the gain, and when V _gain > G _max , the gain V _gain is determined as the _gain to be finally set. To do. Also, V _gain > G
_{When max} does not hold, G _max is determined as the gain to be finally set (step T3).

The offset value is determined in the processing of step T4. As for the offset value R _offset , I _max is AD _max
Set to fit in. Therefore, AD _{max is changed} to I _max
It is divided by the _product of V _gain and V _gain, and that value is determined as R _offset .

The photosensitivity (P
When the image is actually measured using S), the gain and the offset, the actual offset is determined as follows. The maximum voltage that can be input to the offset calculation unit 4 is V
_max , where V '(x, y) is the voltage obtained from a certain point of the sample being obtained, the voltage V (x, y) input to the analog amplifier 5 is V (x, y) = V '(X, y) -V _max (100-R
_offset ) / 100.

According to such an embodiment, the number of scans of the sample is only a predetermined number of times for determining the light receiving sensitivity of the light receiving element 3, and the number of scans for determining the gain and offset can be reduced. The damage to the specimen can be reduced. In addition, the light receiving element 3 is
Since the sensitivity is set according to the effective input range of the / D converter 6 and other parameters are determined, it is possible to prevent the analog light receiving signal from being buried in the dark current of the light receiving element 3.

In the above embodiment, I _max and I _min
I scan the whole screen only once to measure
Only a certain region of interest may be scanned, or the same place may be repeatedly scanned to obtain and calculate the average value of each place.

In this case, if the coordinates of a point on the xy plane are (x, y) and the number of scans on that plane is N,
The average brightness I _ave at that point is expressed by the following equation. I _ave (x, y) = {I ₁ (x ₁ , y ₁ ) + I ₂ (x
₁ , y ₁ ) + ... + I _n (x ₁ , y ₁ )} / N In this I _ave , the maximum and minimum values of brightness are I _max and I
_min .

(Second Embodiment) FIG. 5 shows a system configuration of a scanning microscope using a PMT as a light receiving element. The system configuration is the same as that of the scanning microscope shown in FIG. 1 except that the PMT 3'is used as the light receiving element. Hereinafter, a method of determining the measurement parameters (the applied voltage of the PMT, the offset amount of the offset calculator 4, the gain amount of the analog amplifier 5) set in such a scanning microscope will be described.

FIG. 6 shows the voltage characteristic of the PMT. The characteristic graph shown in FIG. 6 has a logarithmic axis as an axis, the horizontal axis represents the applied voltage (HV), and the vertical axis represents the current multiplication factor. First, the applied voltage (HV) for measuring the dynamic range of the output current of the PMT 3'is determined. Now PMT
3'HV effective range is -500 [V] to -1100
It is assumed that the voltage is [V], the noise such as dark current is small, and the effective voltage value of HV is −700 [V].

The point where the current multiplication factor of this PMT 3'can be set as an integral multiple from the characteristic graph of FIG. 6 is determined within the HV effective range. Here, if it is defined that the current multiplication factor is set in units of 10 times, -520 [V], -600 from FIG.
[V], -700 [V], -1000 [V], -102
0 [V] is selected. Each of these points is called an “HV rank”. The electrical gain should be set to 1 to 10 times. This allows the gain to cover the discrete range of HV.

If the resolution of the A / D converter 6 is 12 bits, the dynamic range can be measured with an accuracy of 4096 gradations. This dynamic range is accurate,
And in order to avoid the influence of noise and to be able to measure,
The HV must be set so that the output current I _max is as close as possible to AD _max and does not exceed AD _max .

The HV rank for determining the dynamic range of the output current is determined according to the flow chart shown in FIG. In the flow chart shown in FIG. 7, the HV rank, which is one rank lower than the HV rank at which the output current I _max overflows AD _max , is selected. In case of overflow, the previous HV rank is adopted. Since the work starting point for HV rank determination is -700 [V], which is the center, the number of repetitions is HV.
Even if the rank is raised to the maximum, it is twice.

The applied voltage corresponding to the HV rank determined as described above is applied to the PMT 3'and the sample is scanned. From the sample image data obtained by this scan, PMT
Measure the dynamic range of the 3'output current.

Next, the gain and offset are determined in the same manner as in the first embodiment according to the flowchart shown in FIG. According to such an embodiment, the number of scans for obtaining the auto gain is less than two, so that even if the subsequent scans for image display are combined, the final image display can be performed by two to three scans. It will be possible. Also. Even when setting parameters on a computer, the hardware module P
The display can be performed without setting MT3 ', the offset calculation unit 4, and the analog amplifier 5. As a result, it is possible to reduce the number of sample scans without forcing the user to perform a troublesome operation.

(Third Embodiment) FIG. 8 shows the system configuration of a scanning microscope in which the intensity of light input to the PMT can be adjusted. The system control computer 7 controls the filter exchange module 11 for inserting and removing the ND filter with respect to the optical path from the light source 1 to the scanning microscope 2. PMT
The configuration until the output of is converted into a digital signal is similar to that of the second embodiment. Hereinafter, a method of determining the measurement parameters (input light amount, offset amount of the offset calculator 4, gain amount of the analog amplifier 5) set in such a scanning microscope will be described.

FIG. 9 shows the relationship between the input light intensity to the PMT and its output current. As shown in the figure, since the input light intensity and the output current of the PMT are linear,
If control is performed at equal intervals between n times, the output current can be set in proportion to 1 to n times.

The rank of the input light (PV) for measuring the dynamic range of the output current from the PMT 3'is determined according to the flowchart shown in FIG. Initial values are set to PV rank = 2, gain = 1, and offset = 0%. The sample is scanned to obtain the maximum value I _max and the minimum value I _min from the image data, and N _over ≦ N _li
If it is _mit , the PV rank is lowered by one rank and the scan is repeated. When N _over > N _limit , the PV rank one rank higher is selected. An instruction is given to the filter exchange module 11 corresponding to the PV rank to set the input light corresponding to the PV rank. N _over > N in the first scan
If it is _limit , the PV rank is increased by one rank and the scan is repeated. When N _over ≦ N _limit , the PV rank is selected. Also at this time, the filter exchange module 11 is instructed to set the input light corresponding to the PV rank.

[0046] By the above processing, PMT3 PV of the input light to the 'maximum value I _max is the closest, and PMT3 controllable so as not to exceed the AD _max in AD _max of the output current of'
The rank has been decided. Here, since the PV rank start point is set to PV rank = 2, even if the PV rank is raised to the maximum, the number of times the scanning is repeated is two.

Next, the gain and offset are determined in the same manner as in the first embodiment according to the flowchart shown in FIG. That is, the gain is calculated so that the dynamic range matches the cover range of the A / D converter. Some samples have gains of 10x and do not subside. In this case, the gain is set to the maximum value of 10 times. The offset is set so that I _max falls within the maximum value AD _max of the A / D converter.

According to such an embodiment, the PMT
The input light to 3'can be set to an optimum value according to the input range of the A / D converter in the subsequent stage, the number of times of scanning the sample can be reduced as in the first embodiment, and the damage to the biological sample can be reduced. Can be made smaller.

In the third embodiment, the input light to the PMT is adjusted by controlling the ND filter, but the same light amount adjustment may be performed by directly controlling the light source by giving a command from the computer. . In this case, the voltage applied to the light source is determined as the measurement parameter. The present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.

[0050]

As described in detail above, according to the present invention, the number of scans required until the measurement parameter is determined is minimized to reduce the damage to the sample, and the parameter setting operation is easy and the user's operation is easy. It is possible to provide a method for determining a measurement parameter that can reduce the burden and can reliably determine the optimum measurement parameter.

[Brief description of drawings]

FIG. 1 is a system configuration diagram of a scanning microscope to which a measurement parameter determination method according to a first embodiment is applied.

FIG. 2 is a diagram showing a relationship between a set sensitivity of a light receiving element and an output current.

FIG. 3 is a flowchart showing a method of setting a rank of light receiving sensitivity according to the first embodiment.

FIG. 4 is a diagram showing a method of setting a gain and an offset in the first embodiment.

FIG. 5 is a system configuration diagram of a scanning microscope to which the measurement parameter determination method according to the second embodiment is applied.

FIG. 6 is a diagram showing a relationship between an applied voltage of PMT and a current multiplication factor.

FIG. 7 is a flowchart showing a method of setting ranks of applied voltages in the second embodiment.

FIG. 8 is a system configuration diagram of a scanning microscope to which the measurement parameter determination method according to the third embodiment is applied.

FIG. 9 is a diagram showing a relationship between input light and output current of a PMT.

FIG. 10 is a flowchart showing a method of setting a rank of an input light quantity according to the third embodiment.

[Explanation of symbols]

1 ... Light source, 2 ... Scanning microscope, 3 ... Light receiving element, 3 '... P
MT, 4 ... Offset calculation unit, 5 ... Analog amplifier, 6
... A / D converter, 7 ... System control computer, 11 ... Filter exchange module.

Claims (3)

[Claims] 1. A laser beam emitted from a laser light source is guided to a focusing optical system via a scanning optical system to scan a sample, and reflected light or transmitted light from the sample scanned by the laser beam is received by a light receiving element. After being incident and converted into an analog electric signal, the analog electric signal is adjusted in offset and gain by an offset adjusting section and an analog amplifying section respectively, and then the signal converted into a digital signal by the A / D converting means is taken in as sample image data, A method for determining measurement parameters in a scanning microscope, which controls measurement parameters such as the light receiving sensitivity of the light receiving element, the offset adjusting section, and the analog amplifying section, offset, and gain from a control computer.
A step of obtaining an input control amount capable of setting the output of the light receiving element in a plurality of stages, (b) a step of (a) in the light receiving element
Step (b) of changing the input control amount until the maximum value of the sample image data satisfies a predetermined condition and repeating step (b). A step of selecting an input control amount suitable for obtaining the dynamic range of the current,
(D) setting the selected input control amount in the light receiving element and measuring the dynamic range of the output current,
(E) A method of determining a measurement parameter for a scanning microscope, comprising the step of determining the offset and gain from the maximum value that can be digitally converted by the A / D conversion means and the dynamic range obtained in step (d). 2. A laser beam emitted from a laser light source is guided through a scanning optical system to a focusing optical system to scan a sample, and reflected light or transmitted light from the sample scanned by the laser beam is converted into a photomultiplier. Is incident on the analog electric signal and converted into an analog electric signal, and the analog electric signal is adjusted in offset and gain by an offset adjusting section and an analog amplifying section respectively, and then the signal converted into a digital signal by A / D converting means is taken in as sample image data. A method for determining measurement parameters in a scanning microscope for controlling measurement parameters such as applied voltage, offset and gain of the photomultiplier, offset adjustment section and analog amplification section from a control computer, and (a) the photomultiplier A step of obtaining an applied voltage capable of setting a current multiplication factor in a plurality of steps, (b) the step Setting the applied voltage obtained in step (a) into the photomultiplier and capturing the sample image data, (c) changing the applied voltage until the maximum value of the sample image data satisfies a predetermined condition, step (b)
Repeatedly selecting the applied voltage suitable for obtaining the dynamic range of the output current of the photomultiplier, and (d) setting the selected applied voltage to the photomultiplier and measuring the dynamic range of the output current. And (e) a step of determining the offset and gain from the maximum value that can be digitally converted by the A / D conversion means and the dynamic range obtained in step (d). . 3. A laser beam emitted from a laser light source is guided to a focusing optical system via a scanning optical system to scan a sample, and reflected light or transmitted light from the sample scanned by the laser beam is received by a light receiving element. After being incident and converted into an analog electric signal, the analog electric signal is adjusted in offset and gain by an offset adjusting section and an analog amplifying section respectively, and then the signal converted into a digital signal by the A / D converting means is taken in as sample image data, A method for determining a measurement parameter in a scanning microscope for controlling measurement parameters such as an input light of the light receiving element, an offset adjusting section and an analog amplifying section, an offset and a gain from a control computer. (A) A plurality of outputs of the light receiving element The step of obtaining the input light intensity that can be set in steps, (b) the input light intensity obtained in step (a) for the light receiving element, And (c) changing the input light intensity until the maximum value of the sample image data satisfies a predetermined condition and repeating step (b). To select an input light intensity suitable for obtaining, (d) setting the selected input light intensity in the light receiving element to measure the dynamic range of the output current, (e) using the A / D conversion means. A method for determining a measurement parameter for a scanning microscope, comprising the step of determining the offset and gain from the maximum value that can be digitally converted and the dynamic range obtained in step (d).