JP4185712B2 - Color microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a color microscope. The present invention is preferably applied to a confocal scanning microscope.
[0002]
[Prior art]
Conventionally, a confocal microscope using a confocal principle is known. This confocal microscope has an objective lens and a pinhole, and when there is a sample at the focal position of the objective lens, the laser beam (response light) that has passed through the pinhole is received by the first light receiving element, so that it is desired to observe Only the image of the height portion (confocal image) is clearly displayed (the resolution is increased). Such a confocal image is a black and white (achromatic) image.
[0003]
However, there is little information in such a black and white video, that is, information on the color of the sample cannot be obtained, and detailed observation such as determination of the type of scratches or deposits may be difficult. For this reason, color (chromatic) microscopes such as those disclosed in Japanese Patent No. 3205530 and Japanese Patent Laid-Open No. 11-14907 have been developed. Generally, this type of color microscope is a light source having a wavelength in the visible region. Is used.
[0004]
[Problems to be solved by the invention]
However, for example, in order to increase the resolution, for example, as a light source (typically a laser light source) for obtaining luminance information, monochromatic light having a wavelength in the short wavelength region (about 400 to 420 nm) in the ultraviolet region or the visible light region. Is used, the wavelength of a light source (typically a white light source) for obtaining color information is significantly different from that of the light source, which causes a problem of chromatic aberration.
[0005]
This chromatic aberration is known as lateral chromatic aberration (magnification chromatic aberration) and vertical chromatic aberration (axial chromatic aberration). When axial chromatic aberration occurs in the objective lens due to light from different light sources, the light source used Depending on the focal length, the focal length varies greatly. For example, since monochromatic light with a short wavelength has a small depth of field, there is a problem in that sufficient luminance information or color information cannot be obtained due to defocusing.
[0006]
Therefore, an object of the present invention is to provide a color microscope that can reduce the influence of axial chromatic aberration of an objective lens caused by light emitted from a plurality of light sources.
[0007]
An object of the present invention is to provide a color microscope capable of obtaining a good screen or image using a plurality of light sources having greatly different wavelengths.
[0008]
A confocal color scanning microscope that can achieve both good resolution and good color reproducibility even when the wavelength of light for obtaining luminance information and the wavelength of light for obtaining color information differ greatly. It is to provide.
[0009]
[Means for Solving the Problems]
  Such a technical problem, according to the first aspect of the present invention,
  Based on information obtained by condensing light from a plurality of light sources through a common objective lens onto a sample, and response light from the sample entering the light receiving means through the common objective lens Color microscope that generates color video signalsIn
  A memory for storing a focal length difference between a focal length of the common objective lens corresponding to one light source of the plurality of light sources and a focal length of the common objective lens corresponding to another light source;
  The plurality of light sourcesThe one light source and the other light sourceTo absorb axial chromatic aberration caused by the light from the lens passing through the objective lensOn the basis of the focal length differenceAdjust the relative distance between the objective lens and the sampleControlThis is achieved by providing a color microscope characterized by having a relative distance adjusting means.
[0010]
According to the second aspect of the present invention, the above technical problem is
Including a first optical system having a first light source for obtaining luminance information and a second optical system having a second light source for obtaining color information, wherein the first and second optical systems are Assuming a color microscope that includes a common objective lens and generates a color video signal by synthesizing the luminance information acquired by the first optical system and the color information acquired by the second optical system,
First focal length measurement means for measuring a focal length of the objective lens in the first optical system;
Second focal length measurement means for measuring the focal length of the objective lens in the second optical system;
The focal length of the objective lens in the first optical system obtained by the first focal length measuring means and the focal length of the objective lens in the second optical system obtained by the second focal length measuring means. A memory for storing the focal length difference between
This is achieved by providing a color microscope having a relative distance adjusting means for adjusting a relative distance between the objective lens and the sample.
[0011]
The present invention is effective when a light source that emits light in a wavelength region in which chromatic aberration correction of a common objective lens used in the first optical system and the second optical system is not sufficiently performed is used. In the present invention, most typically, when the luminance information is obtained by the first optical system or the color information is obtained by the second optical system, prior to this, the objective lens in the first optical system is obtained. Relative distance between the objective lens and the sample by the relative distance adjustment means based on the focal length difference stored in the memory so that the focal length of the objective lens in the second optical system Is adjusted. According to this, when obtaining the luminance information or the color information with the first or second optical system, the luminance information or the color information can be acquired without being affected by the longitudinal chromatic aberration of the objective lens. Therefore, even if the light source used in the first optical system and the light source used in the second optical system are greatly different in wavelength, a good color screen can be obtained.
[0012]
In general, the depth of field by the light source of the second optical system used to acquire color information is larger than the light source of the first optical system used to acquire luminance information. After the color information is acquired once by the two optical systems, a color video signal having no practical problem is generated by synthesizing the luminance information acquired a plurality of times by the first optical system based on the color information. be able to. According to this, since some of the operation steps of the color microscope can be omitted, it is possible to quickly generate a color video signal.
[0013]
The present invention is preferably applied to a confocal color microscope in which the first optical system includes a confocal optical system including a monochromatic light source. In this confocal color microscope, a light source having a wavelength in the ultraviolet region or infrared region can be used as the light source of the first optical system for obtaining luminance information, and particularly in the ultraviolet region or visible region in order to obtain a high-resolution image. Even if a light source that emits monochromatic light having a wavelength in a short wavelength region (for example, about 400 to 420 nm) in the light ray region is employed, a good color screen can be obtained without being affected by axial chromatic aberration.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0015]
1st Embodiment (FIGS. 1-4)
In FIG. 1, a confocal scanning microscope (laser microscope) 100 includes a laser optical system (first optical system) 1 and a white light optical system (second optical system) 2.
[0016]
Laser optical system 1
The laser optical system 1 is a confocal optical system that can detect information relating to the depth of the sample w, and may be laser light having a wavelength in the visible region, but may be laser light in the infrared region or ultraviolet region, preferably the ultraviolet region or visible region. A laser light source 10 that emits laser light L1 having a wavelength in a short wavelength region (about 400 to 420 nm) in the light beam region is provided. In place of the laser light source 10, a light source that emits monochromatic light having a wavelength in the infrared region or the ultraviolet region, for example, a high-intensity lamp may be used, and the laser optical system 1 may be a single one. There may be a plurality. When a plurality of laser optical systems 1 are provided, each laser optical system 1 includes monochromatic light sources having different wavelengths.
[0017]
On the optical axis of the laser light source 10, the first collimator lens 11, the polarization beam splitter 12, the quarter wavelength plate 13, the two-dimensional scanning device 14, the relay lens 15, and the first lens are sequentially arranged from the laser light source 10 side. An imaging lens 16 and an objective lens 17 are provided.
[0018]
A sample stage 30 is disposed in the vicinity of the focal position of the objective lens 17. The sample stage 30 is driven and controlled in the Z direction (up and down direction) by the stage control circuit 40, and manual handles in the X and Y directions. The objective lens 17 focuses the laser beam L1 on the surface of the sample w by adjusting the vertical position of the sample stage 30. The above-described two-dimensional scanning device 14 is composed of, for example, two galvanometer mirrors, and deflects the laser light L1 so that the condensing position on the sample w is two-dimensionally (in the X direction and on the surface of the sample w). (Or Y direction).
[0019]
The sample stage 30 that can be moved up and down adjusts the relative distance between the objective lens 17 and the objective, as shown schematically in phantom lines in FIG. A drive mechanism 31 that moves the lens 17 up and down may be provided.
[0020]
The laser light (response light) L1 reflected by the sample w follows the same path as the laser incident light, passes through the objective lens 17, the first imaging lens 16, and the relay lens 15, and again is the two-dimensional scanning device 14. Through the quarter-wave plate 13 and the polarization beam splitter 12, toward the second imaging lens 18. The laser light L1 that is response light, that is, reflected light, is condensed by the second imaging lens 18 and passes through the optical aperture portion 19a having a pinhole, and the laser light L1 that has passed through the optical aperture portion 19a is the first light receiving element. It is incident on 19b. The first light receiving element 19b is composed of, for example, a photomultiplier or a photodiode, and photoelectrically converts the incident laser light L1 and outputs an analog light quantity signal via an output amplifier and a gain control circuit (not shown). Output to the 1 A / D converter 41.
[0021]
Next, luminance information obtained by the laser optical system 1 will be described.
[0022]
The optical diaphragm 19a is disposed at the focal position of the second imaging lens 18. On the other hand, since the pinhole of the optical diaphragm 19a is very small, the laser beam L1 is focused on the sample w. , The response light L1 is imaged by the second imaging lens 18 at the pinhole of the optical aperture 19a, and the amount of received light incident on the first light receiving element 19b is significantly increased. If L1 is not focused on the sample w, the response light L1 hardly passes through the pinhole of the optical aperture 19a, so that the amount of light received by the first light receiving element 19b is significantly reduced. Therefore, a bright image can be obtained for a focused portion (focused imaging unit) in an imaging region (scanning region) by the laser optical system 1, while a dark image can be obtained for other height portions. It is done. The laser optical system 1 is a confocal optical system that uses the laser light L1 as monochromatic light. In particular, when laser light in the ultraviolet region having a short wavelength is used, luminance information with excellent resolution can be obtained. it can.
[0023]
White light optical system 2
The white light optical system 2 uses a white light source 20 that emits white light (illumination light for color information) L2 as a light source. On the optical axis of the white light source 20, a second collimating lens 21, a first half mirror 22, a second half mirror 23, and the objective lens 17 are disposed. In the first half mirror 22, The white light optical system 2 is arranged so that the optical axes of the two optical systems 1 and 2 coincide. Therefore, the white light L2 passes through the common objective lens 17 and is condensed at the same location as the scanning region of the laser light L1.
[0024]
The white light (response light) L2 reflected by the sample w follows the same path as the white incident light, first passes through the common objective lens 17, and then is reflected by the first half mirror 22, The light is reflected by the second half mirror 23, passes through the third imaging lens 24, and forms an image on the surface of the color CCD (second light receiving element) 25 by the third imaging lens 24. In other words, the color CCD 25 is disposed at a position conjugate to or close to the conjugate with the light diaphragm portion 19a. The image picked up by the color CCD 25 is read as analog color image pickup information to the CCD drive circuit 43 and output to the second A / D converter 42.
[0025]
Here, color imaging information refers to video information consisting of intensities for the three primary colors of light (red, green, and blue), luminance information and color difference information, and composite color video information including horizontal sync signals and color burst signals. This means information that can display a color image as it is or after being processed.
[0026]
Next, the color video signal generating means 5 of FIG. 2 that operates in the color confocal image mode described later will be described.
[0027]
The color video signal creating means 5 creates the digital signals ro, go, bo for color video by combining the luminance information from the first light receiving element 19b and the color information from the color CCD 25. The color video signal generating means 5 includes first and second area circuits 51 and 52, a luminance conversion circuit 53, and the like. Here, luminance information refers to information relating to luminance that does not include color, and “color information” refers to information relating to a balance of color intensity, such as a color difference signal.
[0028]
As shown in FIG. 3, the first and second area circuits 51 and 52 select predetermined common portions of the imaging areas A1 and A2 of the laser optical system 1 and the white light optical system 2 as video areas AO, respectively. The digital signal is output for the selected part. That is, the first area circuit 51 of FIG. 2 stores the luminance signal i in the luminance memory Mi with the resolution corresponding to each pixel of the color CCD 25 for the video area AO. On the other hand, the second area circuit 52 stores red, green, and blue color intensity signals rm, gm, and bm for each pixel in the first color intensity memories Mr1, Mg1, and Mb1 for the video area AO. The color intensity signal is a signal including luminance (intensity) for the three primary colors.
[0029]
The luminance conversion circuit 53 converts the luminance information of the color intensity signals rm, gm, and bm for each pixel into luminance information of the luminance signal i according to the following arithmetic expressions (1), (2), and (3). The conversion color intensity signals ro, go, bo are obtained by replacement, and the signals are stored in the second color intensity memories Mr2, Mg2, Mb2.
[0030]
Ro = I · Rm / (Rm + Gm + Bm) (1)
Go = I · Gm / (Rm + Gm + Bm) (2)
Bo = I · Bm / (Rm + Gm + Bm) (3)
[0031]
Where I is the luminance of the luminance signal i;
Rm, gm, bm are the luminance (intensity) of the color intensity signals rm, gm, bm;
Ro, Go and Bo are the luminances (intensities) of the converted color intensity signals ro, go and bo.
[0032]
The first and second color intensity memories Mr1 to Mb1 and Mr2 to Mb2 have storage units corresponding to the pixels in the above-described video area Ao of the color CCD 25.
[0033]
The converted color intensity signals ro, go, bo obtained in this way are signals obtained by replacing the luminance information in the color imaging information from the color CCD 25 with the luminance information from the first light receiving element 19b. The converted color intensity signals ro, go, bo are read from the second color intensity memories Mr2, Mg2, Mb2 and output to the D / A converter 60. Further, the adder 61 adds the synchronization signal a. Thus, an analog composite color video signal c is obtained. The composite color video signal c is output to the monitor 62, and an image of the sample w is displayed.
[0034]
Method of using scanning microscope 100
The scanning microscope 100 selects and uses one of three modes: an area search mode, a monochrome (achromatic) confocal image mode, and a color confocal image mode (first color slice screen mode). These modes are set by operating the operation unit 63 (FIG. 2).
[0035]
When the area search mode is selected, the color video signal generating means 5 stops the laser drive circuit 44 of FIG. 1 and operates the CCD drive circuit 43 to cause the color CCD 25 to take an image. In this area search mode, the color intensity signals rm, gm, bm stored in the first color intensity memories Mr1, Mg1, Mb1 from the second area circuit 52 in FIG. 2 are output to the D / A converter 60 as they are. A normal enlarged image having a deep depth of field is displayed on the monitor 62. Therefore, by moving the sample stage 30 in FIG. 1 in the X direction and / or the Y direction, it is possible to find an area to be imaged.
[0036]
When the black and white confocal image mode is selected, the color video signal generating means 5 (FIG. 2) operates the laser drive circuit 44 and the two-dimensional scanning device 14 of the laser optical system 1 and causes the laser optical system 1 to take an image. . In this black and white confocal image mode, the luminance signal i stored in the luminance memory Mi from the first area circuit 51 in FIG. 2 is output to the D / A converter 60 as it is, and has a high resolution black and white (achromatic color). Is displayed on the monitor 62.
[0037]
When the color confocal image mode (first slice mode) is selected, the laser drive circuit 44 and the CCD drive circuit 43 are alternately driven as described below.
[0038]
First color slice screen mode (Figure 4)
That is, when the mode is selected in step S1 of FIG. 4, first, the process proceeds to step S2, and the color information is obtained by the white optical system 2 while driving the sample stage 30 in the Z direction (vertical direction) by the stage control circuit 40. The focal length of the objective lens 17 when obtaining the lens, that is, the distance between the objective lens 17 and the sample w is measured in advance, and similarly, the focal length of the objective lens 17 when obtaining the luminance information by the laser optical system 1, ie, the objective. The distance between the lens 17 and the sample w is measured in advance, and the difference between the distances, that is, the focal distance difference r between the white light source 20 and the laser light source 10 is measured in advance. Store in memory. This focal length difference r is substantially an axial chromatic aberration between the white optical system 2 and the laser optical system 1. Instead of driving the sample stage 30, a means for adjusting the relative distance from the sample w may be configured by moving the objective lens 17 up and down by the driving means 31.
[0039]
Next, the process proceeds to step S3, and after one screen is scanned with the laser beam L1, the process proceeds to step S4. In step S4, the laser drive circuit 44 of FIG.
[0040]
Next, in step S5, the sample w is irradiated with the white light source 20. At this time, drive control is performed in the Z direction (vertical direction) by the stage control circuit 40, the distance between the objective lens 17 and the sample w is adjusted, and the focal length previously stored in the memory in step S2 The sample stage 30 is moved upward or downward by a distance corresponding to the difference r. Thereby, the sample w can be positioned at the focal length of the objective lens 17 by the white light source 20, and the light emitted from the white light source 20 is immediately condensed on the sample w. The white light reflected by the sample w accumulates charges in the color CCD 25 (step S6).
[0041]
The color intensity signals rm, gm, bm in FIG. 2 obtained in step S6 are converted by replacing the luminance information contained in the signals with the luminance information of the luminance signal i obtained by the laser beam scanning in step S3. The color intensity signals ro, go and bo. The converted color intensity signals ro, go, bo are respectively stored in the second color intensity memories Mr2, Mg2, Mb2, and then output to the D / A converter 60 to display an enlarged color image on the monitor 62 ( Step S7).
[0042]
Next, in step S8, the stage control circuit 40 controls driving in the Z direction (up and down direction), and moves the sample stage 30 by the same amount in the opposite direction to the previous movement of the sample stage 30 in step S5. In preparation for the next laser beam scanning. That is, until the first color slice image mode is turned off, the operations of steps S3 to S8 are repeated, and the scanning of the laser light L1 and the accumulation and reading of charges by the CCD driving circuit 43 are repeated, so that the color is shared. A focused image is obtained.
[0043]
In the case of the present embodiment, when imaging is performed by the color CCD 25 of FIG. 1 (charge is accumulated in the color CCD 25), the laser driving circuit 44 is stopped so that the laser light L1 does not enter the color CCD 25. Therefore, an image having a color close to the actual color of the sample w can be obtained without forming an image having the color of the laser beam L1.
[0044]
As a means for preventing the laser light L1 from entering the color CCD 25, there are various means such as using a shutter for shielding the laser light L1, or setting the scanning range of the laser light L1 outside the imaging area of the color CCD 25. The method can be adopted. Further, even if the laser beam L1 is incident on the color CCD 25 and takes on the color of the laser beam L1, a color image can be obtained, which is included in the scope of the present invention.
[0045]
5 and subsequent drawings show a modified example of the first embodiment or other embodiments, but the same elements are substantially the same as those included in the first embodiment described above. The description is omitted by attaching the reference numerals.
[0046]
Modification of the first embodiment (FIG. 5)
In the first embodiment described above, by operating the operation unit 63, three modes of the area search mode, the monochrome (achromatic) confocal image mode, and the color confocal image mode (first color slice screen mode) are selected. One of them was selected and used, but the next second color slice screen mode (FIG. 5) was prepared as another additional option or in place of the first color slice screen mode (FIG. 4). May be.
[0047]
That is, when the second color slice screen mode is selected in step S101 of FIG. 5, first, the process proceeds to step S102, and the white optical system 2 performs driving control in the Z direction (vertical direction) by the stage control circuit 40. The focal length of the objective lens 17 when obtaining color information, that is, the distance between the objective lens 17 and the sample w is measured in advance. Similarly, the focal length of the objective lens 17 when obtaining luminance information by the laser optical system 1. That is, the distance between the objective lens 17 and the sample w is measured in advance, and the difference between the distances, that is, the focal distance difference r between the white light source 20 and the laser light source 10 is measured in advance. This is stored in the memory.
[0048]
Next, the process proceeds to step S103, and after the count value (i) is set to zero, in step S104, the operator inputs the number n of luminance information acquisitions for one time of color information acquisition by means of an input unit (not shown). To do. Next, in step S105, after one screen is scanned with the laser beam L1, the process proceeds to step S106. In step S106, the laser drive circuit 44 of FIG.
[0049]
In step S107, the white light source 20 irradiates the sample w. At this time, drive control is performed in the Z direction (vertical direction) by the stage control circuit 40, and the distance between the objective lens 17 and the sample w is set to the focal length difference r previously stored in the memory in step S102. The sample stage 30 is moved upward or downward by a corresponding distance. Thereby, the light emitted from the white light source 20 is immediately collected on the sample w. The white light reflected by the sample w accumulates charges in the color CCD 25 (step S108).
[0050]
The color intensity signals rm, gm, bm in FIG. 2 obtained in step S108 are converted by replacing the luminance information contained in the signals with the luminance information of the luminance signal i obtained by the laser beam scanning in step S105. The color intensity signals ro, go and bo. The converted color intensity signals ro, go, bo are respectively stored in the second color intensity memories Mr2, Mg2, Mb2, and then output to the D / A converter 60 to display an enlarged image of the color on the monitor 62 ( Step S109).
[0051]
Next, in step S110, the stage control circuit 40 controls driving in the Z direction (vertical direction), and the sample stage 30 is moved by the same amount in the opposite direction to the previous movement of the sample stage 30 in step S107. In preparation for the next laser beam scanning.
[0052]
In the next step S111, the count value (i: variable) is incremented to count up the count value. In the next step S112, it is determined whether or not the count value (i) is “i = n”. When is smaller than n, the process proceeds to step S113, where one screen is scanned with the laser beam L1, and the result of this scanning is processed on the monitor 62 and displayed on the monitor 62 (step S114). ). This operation is performed by repeatedly sampling the luminance information for a predetermined number n input by the operator in step S104. During the predetermined number of times of luminance information sampling, the one-time color information obtained in step S108 Displayed in combination.
[0053]
Until the second color slice image mode is turned off (step S115), the operations of steps S105 to 114 are repeated, and the scanning of the laser beam L1 and the accumulation and reading of charges by the CCD driving circuit 43 are repeated. It is.
[0054]
According to the modification of FIG. 5, by using the characteristic that the depth of field when the white light source 20 is used is larger than the depth of field when the laser light source 10 is used, the number of times of obtaining color information is reduced. The update speed of the color slice image of the color scanning microscope 100 can be improved.
[0055]
By the way, when the uneven sample w is observed in the color confocal image mode described above, the image of the portion out of focus (height) is darkened. Therefore, as shown in FIG. 6 and FIG. 7, it is preferable to provide a concave / convex color confocal image (color ultra-deep screen) mode suitable for observation of the concave / convex sample w.
[0056]
When the confocal scanning microscope (laser microscope) 100 is provided with a color confocal image for unevenness (color ultra-deep screen) mode, it is preferable to include a color video signal creating means 5 as shown in FIG. The configuration is the same as that shown in FIG. In other words, the color video signal generating means 5 may have a memory including a maximum luminance storage unit Mmax and a Z coordinate storage unit Mz. The maximum luminance storage unit Mmax stores the maximum luminance Imax received by the first light receiving element 19b (FIG. 2) for each imaging unit (unit corresponding to each pixel of the color CCD 25) in the scanning region. The Z coordinate storage unit Mz stores the Z coordinate (the height of the sample stage 30) when the luminance I reaches the maximum luminance Imax for each imaging unit. The luminance comparison means 54 compares the maximum luminance Imax stored in the maximum luminance storage unit Mmax with the luminance I newly stored in the luminance memory Mi for each imaging unit. As a result of this comparison, if the luminance I newly stored in the memory luminance memory Mi for each imaging unit is larger than the maximum luminance Imax, the luminance I is stored in the maximum luminance storage unit Mmax as the maximum luminance. Mrii, Mgii, and Mbii store the values of rm, gm, and bm at the position obtained by adding γ to the Z coordinate position when the luminance I reaches the maximum luminance Imax. The processing after the luminance conversion circuit 53 is the same as the processing related to the luminance information among the converted color intensity signals ro, go, bo and the color imaging information detailed in the luminance conversion circuit 32 of the first embodiment. Do. The second color intensity memories Mr2, Mg2, and Mb2 constitute a composite imaging information storage unit, and the color imaging received by each pixel of the color CCD 25 at the Z coordinate + γ of each pixel stored in the Z coordinate storage unit Mz. Of the information, a signal obtained by replacing the luminance information with the maximum luminance Imax is stored. The second color intensity memories Mr2, Mg2, and Mb2 store luminance information and color information when the height of the sample stage 30 in FIG. 1 is changed and the first optical system 1 and the second optical system are in focus. The synthesized information is stored for each pixel (imaging unit).
[0057]
First concavity color confocal image (first color ultra-deep screen) mode (FIG. 7)
This first color ultra-deep screen mode is in addition to one of the operation modes such as the region search mode, black and white (achromatic) confocal image mode, first and / or second color slice screen mode described above, When the operator operates the operation unit 63, the first color ultra-deep screen mode can be operated (step S201 in FIG. 7).
[0058]
In FIG. 7, in step S202, the focal length of the objective lens 17 when the white optical system 2 obtains color information while controlling the drive in the Z direction (vertical direction) by the stage control circuit 40, that is, the objective lens 17 and the sample w. In the same manner, the distance between the objective lens 17 and the sample w when the luminance information is obtained by the laser optical system 1, that is, the distance between the objective lens 17 and the sample w is measured in advance. , That is, the difference r in the focal length between the white light source 20 and the laser light source 10 is measured in advance and stored in the memory.
[0059]
Next, after moving the sample stage 30 to the ascending end in step S203, in step S204, imaging is performed by the second optical system 2 (white light source), and charges are accumulated in the color CCD 25 to obtain color imaging information.
[0060]
Subsequently, the process proceeds to step S205 in FIG. 7, and the first optical system 1 scans and images the laser light L1 to obtain luminance information.
[0061]
The luminance information acquired in step S205 is stored in the maximum luminance storage unit Mmax of FIG. 6 for each imaging unit (unit corresponding to the pixel of the color CCD 25), and the Z coordinate at the time of imaging is stored in the Z coordinate storage unit. Store in Mz.
[0062]
Next, the process proceeds to step S206, the sample stage 30 (FIG. 1) is lowered by one step, and then the process proceeds to step S207 in FIG. In this step S207, color imaging information is obtained again by the second optical system 2, and this content is stored for each pixel in the first color intensity memories Mr1, Mg1, Mb1 in FIG. 6, and in step S208 in FIG. move on.
[0063]
In step S208, while scanning the laser beam L1 with the first optical system 1, as described below, the maximum luminance Imax of the maximum luminance storage unit Mmax is updated and stored, and the imaging unit with the updated maximum luminance Imax is updated. The conversion color intensity signals ro, go, bo stored in the second color intensity memories Mr2, Mg2, Mb2 are updated and stored.
[0064]
Next, the process proceeds to step S209 in FIG. 7. If the sample stage 30 is not the descending end + γ, the process returns to step S206. If the sample stage 30 is the descending end + γ, the process proceeds to step S210 and the converted color intensity signal ro. , Go, bo are output to the D / A converter 60, and then a composite color video signal c is obtained. That is, by repeating steps S206 to S209 in FIG. 7, the updating of the maximum luminance Imax and the converted color intensity signals ro, go, bo in the maximum luminance storage unit Mmax in FIG. 6 is repeated. Accordingly, the luminances of the converted color intensity signals ro, go, bo output in step S210 are expressed by the following equations (21)-(23).
[0065]
Ro = Imax.Rm / (Rm + Gm + Bm) (21)
Go = Imax · Gm / (Rm + Gm + Bm) (22)
Bo = Imax · Bm / (Rm + Gm + Bm) (23)
[0066]
If the mode is OFF in step S211 in FIG. 7, the mode is terminated.
[0067]
As described above, in the first color ultra-deep screen mode (first concavity and convexity color confocal image mode), the first optical system 1 and the second optical system 2 are in focus for each pixel of the color CCD 25 in FIG. Since color imaging information about luminance information and color information is used, a focused image can be obtained for each pixel even if there is unevenness, so it is more focused than a normal color enlarged image Video is obtained.
[0068]
By the way, in the first embodiment and the modification thereof described above, the luminance conversion is performed, but this luminance conversion is not always necessary. That is, the color imaging information stored in Mrii, Mgii, and Mbii (FIG. 6) is output to the second color intensity memories Mr2, Mg2, and Mb2 without performing the luminance conversion in Steps S205 and S208 of FIG. That is, the composite color video signal c may be obtained according to the following equations (31) to (33).
[0069]
Ro = Rm (31)
Go = Gm (32)
Bo = Bm (33)
[0070]
Also in this case, as in the modification example illustrated in FIGS. 6 and 7, since color imaging information at the time of focusing on each pixel of the color CCD 25 in FIG. 1 is used, unlike a normal color enlarged image, A strictly focused image can be obtained.
[0071]
Second concavity color confocal image (second color ultra-deep screen) mode (FIG. 8)
This second color ultra-deep screen mode is a modification of the above-described first color ultra-deep screen mode (FIG. 7). Therefore, in addition to one of the operation modes such as the area search mode, the black and white (achromatic) confocal image mode, and the first and / or second color slice screen modes described above, the operator can operate the operation unit 63 (FIG. By operating 2), the second color ultra-deep screen mode can be operated (step S301 in FIG. 8).
[0072]
In the flowchart shown in FIG. 8, substantially the same steps as those in the first color ultra-deep screen mode (FIG. 7) described above are indicated by adding the step numbers in FIG. The description is omitted or incorporated.
[0073]
When the second color ultra-deep screen mode is entered in step S302, the process proceeds to step S302. In step S302, as in step S202 (FIG. 7) in the first mode, the focal length difference r is measured in advance and stored in the memory. Next, the process proceeds to step S303, and in this step S303, the operator inputs the number n of luminance information acquisition times with respect to one acquisition of color information by an input unit (not shown).
[0074]
Next, in step S304, the sample stage 30 is moved to the rising end in the same manner as in step S203 (FIG. 7) in the first mode, and then in step S305, as in step S204 in the first mode. An image is picked up by the optical system 2 (white light source), and charges are accumulated in the color CCD 25 to obtain color image pickup information.
[0075]
Next, the count value (i) is set to zero in step S306, and the process proceeds to the next step S307, and the laser beam L1 is scanned by the first optical system 1 in the same manner as in step S205 (FIG. 7) in the first mode. Then, luminance information is obtained by imaging.
[0076]
Next, the process proceeds to step S308, and in the same manner as step S206 (FIG. 7) in the first mode, after the sample stage 30 (FIG. 1) is lowered by one step, the process proceeds to step S309 and the count value (i) is counted. Up.
[0077]
In the next step S310, as in step S208 in the first mode, while scanning the laser light L1 with the first optical system 1, the maximum luminance Imax of the maximum luminance storage unit Mmax is updated and stored, and the maximum luminance Imax is set. For the updated imaging unit, the converted color intensity signals ro, go, bo stored in the second color intensity memories Mr2, Mg2, Mb2 are updated and stored. That is, the luminance I newly stored in the luminance memory Mi of FIG. 6 by scanning the laser beam L1 and the maximum luminance Imax stored in the maximum luminance storage unit Mmax are compared for each imaging unit. Means 54 compares. If I> Imax as a result of the comparison, the luminance comparison unit 54 stores the luminance I as the new maximum luminance Imax in the maximum luminance storage unit Mmax and the first color intensity memory Mr1, The color information at the maximum luminance position + γ, that is, color imaging information, is converted from the color intensity signals rm, gm, bm stored in Mg1, Mb1, and converted into the second color intensity memory as converted color intensity signals r0, g0, b0. Update and store the addresses of the imaging units of Mr2, Mg2, and Mb2. On the other hand, as a result of the comparison, if the luminance I is equal to or lower than the maximum luminance Imax, the maximum luminance Imax and the converted color intensity signals r0, g0, b0 are not updated for the imaging unit. Since one piece of color imaging information corresponds to n pieces of luminance information, the color imaging information to be adopted is color imaging information at the Z position closest to the maximum luminance + γ.
[0078]
In the next step S311, it is determined whether or not the count value (i) is “i = n”. When the count value is smaller than n, the process proceeds to step S308, and the luminance information is obtained using the same color information. Repeat sampling.
[0079]
In step S311, when the count value (i) is “i = n”, that is, when the sampling of the luminance information of the predetermined number of times input by the operator is completed, the process proceeds to step S312 to check whether the sample stage 30 exists at the lower end. If the sample stage 30 is not the descending end + γ, the process returns to step S305. On the other hand, if the sample stage 30 is the descending end + γ, the process proceeds to step S313 and is the same as step S210 in the first mode. In addition, after the converted color intensity signals ro, go, bo are output to the D / A converter 60, a composite color video signal c is obtained.
[0080]
Second Embodiment (FIG. 9)
In the first embodiment, the laser beam L1 that is focused in a spot shape on the surface of the sample w and the first light receiving element 19b is used. However, the laser beam L1 that is focused on the surface of the sample w and the first light receiving element 19b is linearly focused. The line laser beam L1 may be used. That is, as in the laser microscope 200 of the second embodiment shown in FIG. 9, the line laser beam L1 that is long in the Y direction is used instead of the laser beam L1, and the Y direction is used instead of the dotted first light receiving element 19b. The long one-dimensional CCD 19A is used, and the one-dimensional scanning device 14A is used instead of the two-dimensional scanning device 14. In this case, as shown in FIG. 9B, the line laser light L1 is scanned in a direction orthogonal to the longitudinal direction when the line laser light L1 is collected on the surface of the sample w. The light diaphragm portion 19a has a slit shape (groove shape).
[0081]
In the first embodiment, the white light (response light) L <b> 2 reflected by the sample w is reflected by the first half mirror 22 disposed between the objective lens 17 and the first imaging lens 16. However, in the microscope 200 according to the second embodiment, the second half mirror 23 is formed by sharing the first imaging lens 16 and the third imaging lens 24 of FIG. Is disposed between the first imaging lens 1 and the first relay lens 15.
[0082]
Therefore, the white light (response light) L2 reflected by the sample w passes through the objective lens 17 and the first imaging lens 16, and then is reflected by the second half mirror 22 to be a color CCD (second light receiving element). ) The image is formed on the surface of 25.
[0083]
In the second embodiment, the optical aperture 19a is provided in front of the first light receiving element 19A of the laser optical system 1 in FIG. 9, but the optical aperture 19a is not necessarily provided.
[0084]
Third Embodiment (FIG. 10)
As in the laser microscope 300 of the third embodiment shown in FIG. 10, a monochrome one-dimensional CCD 19A is provided at the focal position of the second imaging lens 18, and the first one-dimensional scanning device 14A is replaced with the first one-dimensional scanning device 14A. A second one-dimensional scanning device 14B may be provided between the quarter-wave plate 13 and the first relay lens 15 between the collimating lens 11 and the polarization beam splitter 12.
[0085]
In the microscope 300 of the third embodiment, the second half mirror 23 may be disposed between the objective lens 17 and the first imaging lens 16 as in FIG. As shown in the figure, it may be arranged between the first imaging lens 16 and the first relay lens 15.
[0086]
Fourth Embodiment (FIG. 11)
A laser microscope 400 of the fourth embodiment shown in FIG. 11 is a combination of the laser microscope 100 of the first embodiment of FIG. 1 and the laser microscope 300 of the third embodiment of FIG. . That is, the optical path configuration from the laser light source 10 to the first relay lens 15 and the optical path configuration from the first relay lens 15 to the first light receiving element 19b are the same as those of the laser microscope 100 of the first embodiment. The optical path configuration between the 1 relay lens 15 and the objective lens 17 is the same as that of the laser microscope 300 of the third embodiment.
[0087]
The preferred embodiment of the present invention has been described above. For example, in the second optical system 2 in FIGS. 1, 9, 10 and 11, the second light receiving element is rotated forward in addition to the color CCD. It may be a secondary black and white surface light receiving element (for example, CCD) using an RGB filter, a dichroic mirror group and three secondary black and white surface light receiving elements for RGB filters, or a MOS. It is also possible to use other solid-state image pickup devices such as a mold or a television camera in which a plurality of image pickup tubes are combined.
[0088]
In the case where the scanning device that scans the laser beam L1 is also used for the white response light, the following example can be considered. In the case of FIG. 11, the two-dimensional scanning device 14 is composed of an X-direction scanning unit and a Y-direction scanning unit (the Y-direction scanning unit is the first relay lens 15 side, in other words, white response light is first scanned in the Y direction. The second half mirror 23 may be disposed between the Y-direction scanning unit and the X-direction scanning unit. In this case, as the second light receiving element, a one-dimensional color light receiving element, a one-dimensional black-and-white light receiving element provided with a rotating RGB filter in front, a dichroic mirror group and three one-dimensional black-and-white light receiving elements are used.
[0089]
In the case of FIGS. 9 and 10, the second half mirror 23 may be disposed between the quarter-wave plate 13 and the one-dimensional scanning device 14A (14B in FIG. 10). In this case, a dichroic mirror group and three one-dimensional black and white light receiving elements are used as the second light receiving elements. Note that a one-dimensional color light-receiving element or a one-dimensional black-and-white light-receiving element provided with a forward RGB filter can be used as the second light-receiving element. In this case, the second light-receiving element can be used as the first light-receiving element. . However, the rotating RGB filter requires a portion (opening or the like) that guides the white response light directly to the one-dimensional monochrome light receiving element in addition to RGB.
[0090]
Similarly, in FIG. 11, the second half mirror 23 may be disposed between the two-dimensional scanning device 14 and the quarter-wave plate 13. In this case, as the second light receiving element, a two-dimensional light receiving element provided with a rotating RGB filter in front, a dichroic mirror group and three two-dimensional light receiving elements are used.
[0091]
In the above-described modification of the arrangement of the second half mirrors 23, the second light receiving element 25, the CCD drive circuit 43, and the second A / D 42 are associated with the change in the arrangement of the second half mirror 23. It goes without saying that the arrangement of the is also changed.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram of a scanning microscope according to a first embodiment.
FIG. 2 is a block diagram.
FIG. 3 is a plan view showing an imaging region.
FIG. 4 is a flowchart for explaining an operation procedure in a first color slice screen mode;
FIG. 5 is a flowchart for explaining an operation procedure in a second color slice screen mode;
FIG. 6 is a block diagram of a color video signal creating unit according to another modification of the first embodiment.
FIG. 7 is a flowchart for explaining an operation procedure in a first color ultra-deep screen mode.
FIG. 8 is a flowchart for explaining an operation procedure in a second color ultra-deep screen mode.
FIG. 9 is a schematic configuration diagram of a scanning microscope according to a second embodiment.
FIG. 10 is a schematic configuration diagram of a scanning microscope according to a third embodiment.
FIG. 11 is a schematic configuration diagram of a scanning microscope according to a fourth embodiment.
[Explanation of symbols]
1: First optical system
16: First imaging lens adjacent to the objective lens
17: Common objective lens
19b: first light receiving element
2: Second optical system
20: Illumination light for color information (white light source)
25: Second light receiving element
31: Driving means for moving the objective lens up and down
40: Sample stage control circuit
L1: Laser light or high-intensity lamp
L2: White light

Claims (8)

Based on information obtained by condensing light from a plurality of light sources through a common objective lens onto a sample, and response light from the sample entering the light receiving means through the common objective lens In color microscopes that generate signals for color images,
A memory for storing a focal length difference between a focal length of the common objective lens corresponding to one light source of the plurality of light sources and a focal length of the common objective lens corresponding to another light source;
The objective lens and the sample based on the focal length difference to absorb axial chromatic aberration caused by light from the one light source and the other light source of the plurality of light sources passing through the objective lens A color microscope having a relative distance adjusting means for adjusting and controlling a relative distance between the two. Including a first optical system having a first light source for obtaining luminance information and a second optical system having a second light source for obtaining color information, wherein the first and second optical systems are In a color microscope that includes a common objective lens and generates a color video signal by combining the luminance information acquired by the first optical system and the color information acquired by the second optical system,
First focal length measurement means for measuring a focal length of the objective lens in the first optical system;
Second focal length measurement means for measuring the focal length of the objective lens in the second optical system;
The focal length of the objective lens in the first optical system obtained by the first focal length measuring means and the focal length of the objective lens in the second optical system obtained by the second focal length measuring means. A memory for storing the focal length difference between
A color microscope comprising: a relative distance adjusting unit for adjusting a relative distance between the objective lens and the sample. Prior to obtaining the luminance information by the first optical system or the color information by the second optical system, the focal length of the objective lens in the first optical system or the second optical system is used. claims of the objective lens focal length so as to on the basis of the focal length difference stored in the memory the relative distance adjusting means and adjusting controlling the relative distance between the sample and the objective lens Item 3. The color microscope according to Item 2. After obtaining the color information by the second optical system once, each time the luminance information is obtained a plurality of times by the first optical system, the obtained luminance information and the color information are combined to be used for color video. The color microscope according to claim 2, further comprising a color video signal generation unit configured to generate the above signal . The first optical system includes a confocal optical system including a monochromatic light source;
The first light source of the first optical system emits visible light, and the second light source of the second optical system emits light having a wavelength different from that of the first light source. The color microscope as described in any one of 2-4. Wherein the wavelength of the first optical system of the light source is a wavelength in the ultraviolet region or infrared region, according to any one of claims 2-4, wherein the second optical system of the light source, characterized in that a white light source Color microscope. The color microscope according to any one of claims 1 to 6, wherein the relative distance adjusting unit includes a unit that moves the objective lens. The color microscope according to any one of claims 1 to 6, wherein the relative distance adjusting unit includes a unit that moves a sample stage on which the sample is placed.

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