JP2861723B2 - Confocal microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a height image calculation of a confocal microscope, and more particularly to a confocal microscope capable of obtaining a high-quality image with less noise.

[0002]

2. Description of the Related Art In recent years, with the improvement of processing technology, micro-
The need for three-dimensional structure measurement is increasing. Also,
In the field of biotechnology, attention has been paid to the three-dimensional structure of molecules. There are interference microscopes, confocal microscopes, and the like that respond to these.

In a confocal microscope, a combination of laser light or non-laser light and a pinhole is used to illuminate at a pinpoint. This prevents scattered light from other than the measurement point. In addition, a pinhole is provided as a spatial filter in front of the light receiver in order to cut off light from points other than the measurement point. Noise light in the same plane as the measurement point forms an image next to the pinhole. Therefore, no light is received by the pinhole. Light at a point shifted in the optical axis direction is spread by the objective lens in front of the pinhole. Therefore,
The light received is drastically reduced. As described above, the confocal microscope can measure only one point in the three-dimensional space. It is also known that this has a resolution in the optical axis direction. This means that the light amount increases only when the focus is on the optical axis, and the light amount becomes almost zero at a point out of focus. At this time, if two-dimensional scanning is performed, a slice image in a three-dimensional space can be measured. Further, since the amount of light peaks at the focal position, in a sample having a surface such as a semiconductor, the optical axis position where the amount of light is maximum is considered to be the surface of the sample. Therefore,
By performing a peak search for each pixel in the plurality of slice images at the position where the maximum light amount is provided in the optical axis direction, the “height at which the peak value of each pixel is given” is set as “the surface of the sample”.
A three-dimensional surface shape image is known.

[0004]

However, if the sample has a steep slope or no peak during scanning, the "surface" does not originally appear in the pixel. However, if the peak search is performed unconditionally, "noise" will eventually be picked up and the image quality will be degraded.

The present invention has been made in view of the above-mentioned problems of the prior art. Pixels that do not make sense even when a peak search is performed are excluded so that good image quality free from noise can be obtained. It is intended to provide a focusing microscope.

[0006]

According to the present invention, there is provided a confocal arrangement for rotating a pinhole substrate and scanning a sample with irradiation light passing through the pinhole substrate. In a confocal microscope using an optical scanner, a confocal microscope that obtains a three-dimensional image or an image with a deep depth of focus by mutually calculating slice images obtained by confocal imaging. When the brightness is less than a specified value, the corresponding pixel output value (height or brightness)
Is output as zero or an interpolated value from surrounding pixels. In the above calculation, a value of an image when confocal illumination is not performed is subtracted from each confocal image and output.

[0007]

According to the present invention, a pixel that is not a true surface is detected from its brightness itself, and is subjected to an exclusion process.
High quality image data is obtained.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to the drawings. FIG. 1 shows an embodiment of a configuration for performing a height image calculation of a confocal microscope according to the present invention. The configuration of the configuration is the same as that of a conventional apparatus. In FIG. 1, 1 is a light source, 2 is a confocal optical scanner, 3 is an objective lens, 4 is a sample, and 5 is a sample 4
A Z stage that scans in the (height) direction, 6 is an A / D converter, and 7 is a CPU that measures and calculates a plurality of slice images while moving the Z stage 5 up and down. 8 is a memory,
Reference numeral 9 denotes a frame memory, and 10 denotes a display unit. The calculation result of the CPU 7 is monitored by the display unit 10 through the frame memory 9.

The operation of such a configuration will be described below. The present invention relates to the measurement and calculation of the height image by the CPU 7, and the operation of the optical system is the same as the conventional one. The description is omitted, and only the height image measurement / calculation operation in the CPU 7 according to the present invention will be described with reference to the flowchart shown in FIG. Note that FIG.
Symbols used in the following are a (i, j, k): data of a slice image p (i, j): brightness of a pixel when the light amount is the maximum h (i, j): light intensity of the pixel at the maximum The height number of the Z stage 5 at the time i, j: x, y coordinates of a pixel (i = 1, 2, ──, _Ni , j = 1, 2, ──, N _j ) k: Height of the Z stage The number (k = 1, 2,..., N _k ).

Returning to FIG. 2, first, the slice image data a, the brightness p of the pixel when the light amount is maximum, and the height number h of the stage when the light amount is maximum are set to 0, respectively. Next, Z
It is determined whether the height number k of the stage 5 is k = _Nk , that is, whether it is the maximum value. Here, when the height number k ≠ N _k of the Z stage 5, the slice image data a (i, j, k) having the height number k of the Z stage 5 is measured, and the slice image data a (i , J, k) and the brightness p (i, j) of the pixel when the light quantity is the maximum, and if a> p, the slice image data a is converted to the brightness p of the pixel when the light quantity is the maximum. , the amount of light is substituted respectively the height number k of the Z stage 5 to the height number h of stages at the maximum, as the height number k = k + 1 of the Z stage 5, the flow returns to k = N _k determination of whether. This operation is repeatedly performed until k = N _k .

Next, if the height number k of the Z stage 5 is k = N _k , the brightness p (i,
j) is smaller than a certain value (noise level) b, the pixel brightness p (i, j) = 0 when the light amount is the maximum, and the stage height number h (i, j) when the light amount is the maximum j) = 0.

That is, when the confocal light does not return accurately, the light quantity becomes much darker than other measurement points. For this reason, if it is darker than the noise level b, it is considered that the peak search is meaningless. Therefore, by forcibly setting the corresponding pixel to zero, only a true confocal image can be obtained, and good image quality can be obtained. be able to.

If the observation is made with a normal optical microscope before the confocal measurement, the background light is also picked up. In this case, the data c (i, j) of the image before the confocal illumination is started is measured and subtracted from each of the confocal slice images a (i, j, k). Just do it.

The pixel determined not to be confocal may not be set to zero, but may be used as an interpolation value obtained from pixels before, after, right and left of the pixel.

[0015]

As described above in detail with the embodiment, according to the present invention, a pixel having no meaning even when a peak search is performed is excluded to obtain a good image quality free from noise. Confocal microscope can be realized.

[Brief description of the drawings]

FIG. 1 is a configuration example for performing a height image calculation of a confocal microscope according to the present invention.

FIG. 2 is a flowchart for explaining the operation of the present invention.

[Explanation of symbols]

 2 Optical scanner for confocal 4 Sample 5 Z stage 7 CPU

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Claims (2)

(57) [Claims] 1. A confocal microscope using a confocal optical scanner that rotates a pinhole substrate and scans a sample with irradiation light passing through the pinhole substrate, wherein a confocal slice image is provided. In a confocal microscope that obtains a three-dimensional image or an image with a deep depth of focus by mutually computing, when the maximum brightness of the pixel is equal to or less than a specified value, the value of the corresponding pixel output is obtained. A confocal microscope wherein (height or luminance) is set to zero or an interpolation value from surrounding pixels and output. 2. The confocal microscope according to claim 1, wherein a value of an image when confocal illumination is not performed is subtracted from each confocal image and output during the calculation. microscope.