JP2004212622A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscope realizing the effective utilization of a part with non-linear distortion near both ends of a scanning range by a resonate type scanner and reciprocative scanning. <P>SOLUTION: In an optical scanner for scanning the surface of a sample with light, a horizontal scanner is constituted to use the resonate type scanner, light receiving information obtained by receiving the light from the sample scanned with the light by the resonate type scanner by a photodetector is sampled every fixed time and stored in a buffer memory 70, and the sampling data stored in the buffer memory 70 are read out at unequal interval addresses according to an address correspondence table 73 obtained by arithmetic processing including inverse trigonometric function arithmetic operation, whereby the received light information at every pixel position at nearly equal intervals on the surface of the sample in a measuring range is obtained. In the case of reciprocative scanning, the order of addresses for reading out the sampling data is set to reverse directions between forward direction scanning and reverse direction scanning. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention scans a sample surface within a measurement range determined by an optical magnification with light, receives light from the sample with a light receiving element via a confocal optical system, and adjusts the height of the sample surface based on the received light information. The present invention relates to a confocal microscope for acquiring distribution information or image information, and more particularly, to a confocal microscope in which at least a part of an optical scanning unit is configured by a resonance type scanner.
[0002]
[Prior art]
In measurement of a sample using a confocal microscope, the surface of the sample is scanned with light (for example, laser light) by an optical scanning unit. Light from the sample (for example, reflected light) is received by a light receiving element via a confocal optical system, and based on the amount of received light, information such as height distribution information of the sample and an ultra-deep image (an image having a very deep focal depth) is obtained. Image information is obtained. When the distance between the sample placed on the stage and the objective lens is changed in the direction of the optical axis, the intensity (light receiving amount) of light incident on the light-receiving element through the pinhole of the confocal optical system changes. When the surface is focused, the amount of received light is maximized. Therefore, height information of the surface of the sample is calculated from the distance information between the sample and the objective lens when the maximum amount of received light is obtained, and the height distribution of the surface of the sample is obtained by scanning the surface of the sample with light. be able to.
[0003]
Also, by combining information on the amount of light received when each point (pixel) on the sample surface is in focus (that is, information on the maximum amount of light received by each pixel), a monochrome image of the sample surface with a very deep depth of focus can be obtained. Obtainable. This image is a so-called super-depth image. Alternatively, if the distance between the sample and the objective lens when the maximum amount of received light is obtained at an arbitrary pixel of interest is fixed, the amount of light received by the pixel at the portion where the height difference from the portion of the pixel of interest is large becomes extremely small. An image (so-called slice image) in which only the portion having the same height as the pixel is bright can be obtained.
[0004]
The optical scanning means of the confocal microscope as described above is usually a two-dimensional optical scanning means comprising a horizontal scanning means and a vertical scanning means. In this case, a galvano (electromagnetic) scanner is used for the vertical scanning means having a relatively long scanning cycle (low frequency), and a resonant (resonant) scanner is used for the horizontal scanning means having a short scanning cycle (high frequency). Often used.
[0005]
While a galvano scanner can change the angle of a mirror at approximately the same speed, a resonant (resonant) scanner changes the angle of the mirror by vibration using a sine wave as a drive signal, so the angular velocity is sinusoidal. , And becomes maximum near the center of the scanning range, and the angular velocity approaches zero as approaching both ends of the scanning range. Therefore, a dimensional difference in the scanning direction per pixel between the center and both ends of an image obtained by optical scanning appears as image distortion. That is, distortion occurs in which the central portion of the image is extended and the peripheral portion is blocked.
[0006]
In order to suppress this image distortion, it is necessary to use a portion near the center of the scanning range of the resonance type scanner where the angular velocity changes relatively linearly, that is, a portion which changes substantially linearly around the zero cross of the sine wave. There is. However, it is necessary to secure a certain adjustment area outside the area actually used, such as in the case of performing offset adjustment for alignment with a color image obtained by a color image sensor. For this reason, it is advantageous to be able to effectively use a region as large as possible including a portion with nonlinear distortion near both ends in the scanning range of the resonance type scanner.
[0007]
Also, due to the characteristics of the resonance type scanner, the time required for the forward path and the time required for the return path are the same. Therefore, when only the outward path is used for scanning, only half of one cycle is used, and the other half is wasted time. Therefore, it is conceivable to perform reciprocal scanning that also effectively uses the return path.
[0008]
As described above, as a conventional technique for effectively utilizing a portion with non-linear distortion near both ends of the scanning range of the resonance type scanner, an auxiliary laser beam is applied to the back surface of the mirror of the resonance type scanner. There is a method of sampling a sample at non-equidistant timing obtained by scanning a pattern at regular intervals with reflected light (see, for example, JP-A-2000-147395). At this time, by reversing the arrangement order of the sampling data on the return path using the line memory, reciprocal scanning becomes possible.
[0009]
[Problems to be solved by the invention]
However, the conventional method as described above requires an optical system for irradiating the back surface of the mirror of the resonance type scanner with auxiliary laser light and scanning the equally-spaced pattern with the reflected light, and the structure is complicated accordingly. become. In addition, precision processing technology is required.
[0010]
The present invention has been made in view of the above-described problems, and effectively utilizes a portion of the scanning range of the resonance type scanner having nonlinear distortion near both ends thereof without adding a new optical system, and performs reciprocating scanning. It is an object to provide a confocal microscope that has been made possible.
[0011]
[Means for Solving the Problems]
The confocal microscope of the present invention scans a sample surface within a measurement range determined by optical magnification with light, receives light from the sample with a light receiving element via a confocal optical system, and based on the received light information, the sample surface. A confocal microscope for acquiring height distribution information or image information, wherein at least a part of light scanning means for scanning a sample surface with light is configured using a resonance type scanner, and the resonance type scanner The light-receiving information obtained by receiving light from the scanned sample by the light-receiving element is sampled at predetermined time intervals, stored in a buffer memory, and according to an address correspondence table obtained by an arithmetic process including an inverse trigonometric function operation. By reading out the sampling data stored in the buffer memory at non-equidistant addresses, each pixel position at substantially equal intervals on the sample surface within the measurement range is read. And obtaining the reception information.
[0012]
According to such a configuration, unequally-spaced sampled data sampled at regular time intervals is obtained by an address conversion process using a buffer memory, instead of a method of adding an optical system as described in the related art. Can be converted into the light reception information of the pixel position. Thus, it is possible to eliminate the distortion in which the central portion of the generated image is extended and the peripheral portion is blocked.
[0013]
In a preferred embodiment, in order to realize the reciprocating scanning by the resonance type scanner, in the address correspondence table, the order of the address for reading out the sampling data in the forward scanning and the order of the address for reading out the sampling data in the backward scanning are set in opposite directions. I have to.
[0014]
In another preferred embodiment, when the sampling data is read from the buffer memory at non-equidistant addresses according to the address correspondence table, the interpolation processing is performed by using the sampling data of two adjacent addresses, so that the pixels at substantially equal intervals are read. Increase the accuracy of the received light information of the position. That is, since the addresses corresponding to the equally-spaced pixel positions obtained by the inverse trigonometric function operation are values including fractions after the decimal point, interpolation processing is performed using sampling data of two adjacent addresses using the fractions as interpolation coefficients. By doing so, the accuracy is increased.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0016]
FIG. 1 shows a schematic configuration of a confocal microscope system according to an embodiment of the present invention. The confocal microscope system 1 includes a confocal microscope having a confocal optical system 2 and a non-confocal optical system 3, a laser driving circuit 44, a first AD converter 41, a CCD driving circuit 43, and a second AD converter of the confocal microscope. 42, a controller including an objective lens moving mechanism 40, a controller 46 using a microcomputer, and the like, and a display device 47 and an input device 48 connected to the controller.
[0017]
First, the confocal optical system 2 of the confocal microscope and its signal processing will be described. The confocal optical system 2 includes a light source 10 for irradiating the sample wk with monochromatic light (for example, laser light), a first collimating lens 11, a polarizing beam splitter 12, a quarter-wave plate 13, a horizontal scanning device 14a, and vertical scanning. The device 14b includes a first relay lens 15, a second relay lens 16, an objective lens 17, an imaging lens 18, a pinhole plate 9, a light receiving element 19, and the like.
[0018]
As the light source 10, for example, a semiconductor laser that emits blue laser light is used. Laser light emitted from the light source 10 driven by the laser drive circuit 44 passes through the first collimator lens 11, the optical path is bent by the polarization beam splitter 12, and passes through the 波長 wavelength plate 13. Then, after being deflected in the horizontal (horizontal) direction and the vertical (vertical) direction by the horizontal scanning device 14a and the vertical scanning device 14b, the light passes through the first relay lens 15 and the second relay lens 16, and is sampled by the objective lens 17. The light is focused on the surface of the sample wk placed on the stage 30.
[0019]
The horizontal scanning device 14a is configured by a resonant (resonant type) scanner, and the vertical scanning device 14b is configured by a galvano (electromagnetic) scanner. By deflecting the laser light in both the horizontal and vertical directions, the surface of the sample wk is scanned with the laser light. For convenience of description, the horizontal direction is referred to as an X direction, and the vertical direction is referred to as a Y direction. The objective lens 17 is driven in the Z direction (optical axis direction) by the objective lens moving mechanism 40. Thereby, the distance in the optical axis direction between the focus of the objective lens 17 and the sample wk can be changed.
[0020]
However, the distance in the optical axis direction between the focal point of the objective lens 17 and the sample wk can be changed by another method. For example, instead of driving the objective lens 17 in the Z direction, the sample stage 30 may be driven in the Z direction. Alternatively, a configuration in which the focal point of the objective lens 17 is moved in the Z direction by inserting a lens whose refractive index changes between the objective lens 17 and the sample wk is also possible. The sample stage 30 can be displaced in the X, Y, and Z directions by manual operation.
[0021]
In the confocal microscope of the present embodiment, the objective lens 17 can be electrically moved in the Z-axis direction via the objective lens moving mechanism 40 by a control signal from the control unit 46, and the sample stage 30 is provided with a stage manual operation mechanism. It can be displaced in the X direction, the Y direction and the Z direction by a manual operation via 31. In addition, the objective lens 17 can be moved up and down via the control unit 46 and the objective lens moving mechanism 40 by a key operation of the input device 48 (for example, an operation of an up / down key).
[0022]
The laser light reflected by the sample wk reverses the above optical path. That is, the light passes through the objective lens 17, the second relay lens 16, and the first relay lens 15, and again passes through the quarter-wave plate 13 via the horizontal scanning device 14a and the vertical scanning device 14b. As a result, the laser light passes through the polarization beam splitter 12 and is condensed by the imaging lens 18. The condensed laser light passes through the pinhole of the pinhole plate 9 disposed at the focal position of the imaging lens 18 and enters the light receiving element 19. The light receiving element 19 is composed of, for example, a photomultiplier tube (photomultiplier tube) or a photodiode, and converts the amount of received light into an electric signal. The electric signal corresponding to the amount of received light is provided to the first AD converter 41 via an output amplifier and a gain control circuit (not shown), and is converted into a digital value.
[0023]
The height (depth) information of the sample wk can be acquired by the confocal optical system 2 configured as described above. The principle will be briefly described below.
[0024]
As described above, when the objective lens 17 is driven in the Z direction (optical axis direction) by the objective lens moving mechanism 40, the distance in the optical axis direction between the focus of the objective lens 17 and the sample wk changes. When the focal point of the objective lens 17 is focused on the surface of the sample wk, the laser light reflected on the surface of the sample wk is condensed by the imaging lens 18 via the above optical path, and almost all the laser light is It passes through the pinhole of the pinhole plate 9. Therefore, at this time, the amount of light received by the light receiving element 19 is maximized. Conversely, when the focus of the objective lens 17 is shifted from the surface of the sample wk, the laser light condensed by the imaging lens 18 focuses on a position shifted from the pinhole plate 9, so that a part of the laser beam is focused. Only light can pass through the pinhole. As a result, the amount of light received by the light receiving element 19 is significantly reduced.
[0025]
Therefore, if the amount of light received by the light receiving element 19 is detected at any point on the surface of the sample wk while driving the objective lens 17 in the Z direction (optical axis direction), the objective lens 17 when the amount of received light is maximized is detected. (Distance in the optical axis direction between the focal point of the objective lens 17 and the sample wk) can be uniquely obtained as height information.
[0026]
Actually, each time the objective lens 17 is moved by one step (one pitch), the horizontal scanning device 14a and the vertical scanning device 14b scan the surface of the sample wk to obtain the amount of light received by the light receiving element 19. When the objective lens 17 is moved in the Z direction from the lower end to the upper end of the measurement range in the Z direction, light reception amount data that changes according to the Z direction position is obtained for each point (pixel) in the scanning range.
[0027]
FIG. 2 is a graph showing an example of received light amount data that changes according to the Z-direction position of the objective lens 17. Based on such received light amount data, the maximum received light amount and the Z-direction position at that time are obtained for each point (pixel). Therefore, the distribution of the surface height of the sample wk on the XY plane is obtained.
[0028]
The obtained surface height distribution information can be displayed on the monitor screen of the display device 47 by several methods. For example, the height distribution (surface shape) of the sample can be displayed three-dimensionally by three-dimensional display. Alternatively, the height data can be converted into luminance data to be displayed as a two-dimensional distribution of brightness. By converting the height data into color difference data, the height distribution can be displayed as a color distribution.
[0029]
Further, a surface image (monochrome image) of the sample wk can be obtained from a luminance signal using the received light amount obtained for each point (pixel) in the XY scanning range as luminance data. If a luminance signal is generated using the maximum amount of received light in each pixel as luminance data, an ultra-deep image with an extremely deep focal depth focused at each point having a different surface height can be obtained. When the height (Z direction position) at which the maximum amount of received light is obtained at an arbitrary target pixel is fixed, the amount of received light of a pixel having a large height difference from the target pixel becomes extremely small. A bright image is obtained only in the portion having the same height as.
[0030]
Next, the non-confocal optical system 3 provided in the confocal microscope and its signal processing will be described. The non-confocal optical system 3 includes a white light source 20, a second collimating lens 21, a first half mirror 22, a second half mirror 23, and a color for irradiating the sample wk with white light (illumination light for capturing a color image). It includes a CCD (image sensor) 24 and the like. In addition, the non-confocal optical system 3 shares the objective lens 17 of the confocal optical system 2, and the optical axes of the two optical systems 1 and 2 partially match.
[0031]
For example, a white lamp is used as the white light source 20, but natural light or indoor light may be used. The white light emitted from the white light source 20 passes through the second collimating lens 21, the optical path is bent by the first half mirror 22, and is focused on the surface of the sample wk placed on the sample stage 30 by the objective lens 17. .
[0032]
The white light reflected by the sample wk passes through the objective lens 17, the first half mirror 22, and the second relay lens 16, is reflected by the second half mirror 23, enters the color CCD 24, and forms an image. The color CCD 24 is provided at a position conjugate with or close to the pinhole of the pinhole plate 9 of the confocal optical system 2. The color image picked up by the color CCD 24 is read out by the CCD drive circuit 43, and its analog output signal is given to the second AD converter 42 and converted into a digital value. The color image thus obtained is displayed on the monitor screen of the display device 47 as an enlarged color image for observing the sample wk.
[0033]
In addition, the super-depth image obtained by the confocal optical system 2 and the normal color image obtained by the non-confocal optical system 3 are combined, and a deep color super-depth having a focal depth substantially in focus at all pixels. Images can also be generated and displayed.
[0034]
The controller including the control unit 46 is also in charge of the processing regarding the color image as described above. An input device 48 such as a console (operation console) and a display device 47 such as a CRT (cathode ray tube) or an LCD (liquid crystal display device) are connected to the controller. A pointing device such as a mouse is also connected as the input device 48.
[0035]
The user can set various measurement parameters using the input device 48 according to the guidance displayed on the screen of the display device 47. For example, a moving range (measurement range) and a moving pitch of the objective lens 17 in the Z direction are set. Alternatively, by setting the light receiving sensitivity (PMT gain) of the light receiving element 19 and the amount of attenuation by the ND filter according to the light reflectance of the surface of the sample wk, etc., the super-depth image or the slice image displayed on the screen is appropriate. Adjust so that the brightness (brightness) is high. Further, the shutter speed, gain, and white balance for obtaining a color image by the color CCD 24 are set. In addition, various scan modes are set as described later.
[0036]
Further, the confocal microscope system 1 (controller thereof) of the present embodiment is also provided with an interface for connecting an external computer system such as a personal computer. By connecting the external computer system with the dedicated software installed to the confocal microscope system 1, the above-described measurement conditions are set on the screen of the external computer system, or the processing of images acquired by the confocal microscope system 1 is performed. Can be displayed on the screen of the external computer system and processed.
[0037]
FIG. 3 is a block diagram illustrating an example of a hardware configuration in which an external computer system 50 is connected to a controller of the confocal microscope system 1. The external computer system 50 includes a display device 51 such as a CRT or LCD, a keyboard 52, a mouse (or another pointing device) 53, a communication interface 54 such as an RS232C, a USB (universal serial bus), IEEE1394, and a processing device (CPU). 55, a main memory 56 as a semiconductor storage medium, a fixed disk device 57 as an auxiliary storage device, and a removable disk device 58.
[0038]
The dedicated software for controlling the confocal microscope system 1 is supplied in a state stored in a storage medium 59 such as a CD-ROM, and is supplied from the storage medium 59 by a removable disk device 58 such as a CD-ROM drive. It is read and installed in the fixed disk device 57. The program installed in the fixed disk device 57 is loaded into the main memory 56 and executed by the processing device 55.
[0039]
The processing executed by such dedicated software includes processing for setting measurement conditions of the confocal microscope system 1 and processing of an image obtained as a result of the measurement. Next, the selection of the scanning mode in the screen display for setting the measurement conditions will be described.
[0040]
FIG. 4 is a diagram illustrating an example of a screen display for setting measurement conditions including selection of a scanning mode. In a screen display 60 displayed on the display device 51, a left area 61 is obtained from a color image obtained from the color CCD 24 of the non-confocal optical system 3 of the confocal microscope system 1 or from a light receiving element 19 of the confocal optical system 2. An area for displaying the obtained confocal image or the measurement result of the height distribution and the like, and on the right side thereof, a vertically long area 62 for setting measurement conditions is displayed.
[0041]
FIG. 5 is an enlarged view of an area 62 for setting measurement conditions in the screen display 60 of FIG. A selection block 63 for selecting a scanning mode is provided on the right side of the portion where “scan setting” is displayed. When the downward triangle mark on the right end of the selection block 63 is clicked with the mouse 53, a pull-down menu 64 as shown in FIG. 5 appears. The pull-down menu 64 includes five types of scanning modes “Normal”, “Part 1/2”, “Part 1/3”, “Skip”, and “Skip 1/2” as options. One scanning mode can be selected from these options using the mouse 53.
[0042]
“Normal” is a normal scanning mode in which the scanning range is the entire measurement range on the XY plane determined by the optical magnification and the number of scanning lines is the maximum number (for example, 768). In "Part 1/2", the vertical scanning range becomes 1/2 of the central portion of the whole, and the number of scanning lines also becomes 1/2 (for example, 384) accordingly. In “Part 1/3”, the vertical scanning range becomes 1/3 of the central portion of the whole, and the number of scanning lines becomes 1/3 accordingly.
[0043]
In “skip”, the scanning range is the entire measurement range, but the number of scanning lines is halved. That is, the interval between adjacent scanning lines is doubled. In the “skip 1/2”, the interval between adjacent scanning lines is doubled, and the vertical scanning range is the central half of the whole. Therefore, the number of scanning lines is reduced to 1/4. Note that the horizontal scanning range does not change in any of the scanning modes and is equal to the horizontal length of the entire measurement range. If the interval between adjacent scanning lines is doubled due to skipping, pixel data between the scanning lines is generated by interpolation.
[0044]
In FIG. 5, although partially hidden by the pull-down menu 64, "double scan" is displayed below the portion where "scan setting" is displayed, and a check box 65 is provided on the left side. When the user checks this check box 65 with the mouse 53, reciprocal scanning is set for horizontal scanning, and when unchecked, normal one-way scanning is set.
[0045]
As described above, the horizontal scanning device 14a for horizontal scanning is constituted by a resonant (resonant type) scanner, and the reflection surface of the mirror vibrates at a predetermined frequency in a self-excited manner by a sine wave drive signal. Therefore, the time required for the forward path and the return path are the same (half cycle) and the phase changes are the same, so that reciprocal scanning is possible. In the case of performing reciprocal scanning, if the number of horizontal scans per vertical scan is the same as in the case of one-way scanning, apparently twice the number of scanning lines can be obtained, and the measurement accuracy is improved accordingly. Become. Conversely, even if the number of horizontal scans per vertical scan is reduced by half, the apparent number of scan lines is the same, so that the measurement accuracy hardly decreases. If the number of horizontal scans per vertical scan is reduced by half, the time required for the entire measurement is reduced by nearly half.
[0046]
However, since the resonance type scanner changes the angle of the mirror by vibration using a sine wave as a drive signal, the angular velocity changes like a sine wave, becomes maximum near the center of the scanning range, and becomes closer to both ends of the scanning range. Approaches zero. Therefore, a dimensional difference in the scanning direction per pixel between the center and both ends of an image obtained by optical scanning appears as image distortion. That is, distortion occurs in which the central portion of the image is extended and the peripheral portion is blocked.
[0047]
As a method for eliminating this image distortion, in the confocal microscope system 1 of the present embodiment, the received light information is sampled at regular time intervals and stored in a buffer memory, and the address correspondence obtained by an arithmetic processing including an inverse trigonometric function operation is calculated. The sampling data stored in the buffer memory is read out at non-equidistant addresses in accordance with the table, thereby obtaining light receiving information at substantially equally spaced pixel positions on the sample surface within the measurement range.
[0048]
FIG. 6 is a block diagram showing a configuration for obtaining received light amount data at equally spaced positions from sampling data of received light amounts obtained by scanning using a resonance scanner in the present embodiment. The voltage signal output from the light receiving element 19 is converted into a digital value by the first AD converter 41. At this time, a digital value is input to the buffer memory 70 as sampling data at predetermined time intervals according to a timing signal given from the timing generator 71. The buffer memory 70 stores the input sampling data at regular intervals in the order of addresses.
[0049]
The read address generator 72 performs addressing of the buffer memory 70 in accordance with an address correspondence table 73 described later, so that (part of) the data stored in the buffer memory 70 is read at non-equidistant addresses. As a result, the received light amount data for each pixel position at equal intervals is read. As will be described later, the address correspondence table 73 is obtained by an arithmetic processing including an inverse trigonometric function operation at the stage when the measurement condition including the scanning mode is set, and is stored in the memory.
[0050]
The data read out from the data stored in the buffer memory 70 at the non-equidistant addresses may be used as it is as the received light amount data for each of the equally-distant pixel positions. However, in the configuration shown in FIG. As a result, the accuracy of the received light amount data for each pixel position at equal intervals is improved. In other words, since the addresses corresponding to the equally-spaced pixel positions obtained by the inverse trigonometric function operation are values including fractions after the decimal point, interpolation processing is performed using sampling data of two adjacent addresses using the fractions as interpolation coefficients. By doing so, the accuracy is increased. Details of this interpolation calculation will be described later.
[0051]
When the reciprocating scanning mode is selected when the address correspondence table 73 is generated, the order of the address for reading the sampling data in the forward scanning and the order of the address for reading the sampling data in the reverse scanning are set in opposite directions. I have to.
[0052]
Next, the address correspondence table 73 will be described. FIG. 7 is a graph showing a change in the position of the scanning spot (light spot) by the resonance type scanner (more precisely, a change in the angle of the mirror). For example, when the time changes from t0, t1,... T14, it is assumed that the position of the light spot changes from the left end (+ D) to the right end (-D). Of course, the actual sampling time is finer.
[0053]
Since the angle of the mirror changes sinusoidally in the resonance type scanner, the position of the scanning spot corresponding to the equally spaced times t0, t1,... T14 is, as shown in FIG. , And become unequally spaced at both ends. Therefore, if data of the amount of received light sampled at regular intervals is used as it is as pixel data at equal intervals, a distortion occurs in which the central portion of the image is extended and the peripheral portion is clogged. FIG. 7B shows a state in which the times t0, t1,... T14 are non-equidistant when the positions of the scanning spots are made equidistant.
[0054]
7A and 7B, when the position of the scanning spot is P, the scanning period is T, and the times t0, t1,... T14 are represented by a variable t, the trigonometric function (cosine function) cos If used, P = Dcos (2πt / T). If an inverse trigonometric function (inverse cosine function) arccos is used, t = (T / (2π)) arccos (P / D). For example, the time t0 corresponding to the position of P = + D (left end) is (T / (2π)) arccos (1) = 0, and the time t14 corresponding to the position of P = −D (right end) is (T / (2π )) Arccos (-1) = T / 2. In general, when a moving pitch (corresponding to a pixel pitch) of a position of a scanning spot is a and n is a natural number, a time t (n) corresponding to a position of P = D (1-an) is t (n). ) = (T / (2π)) arccos (1-an).
[0055]
Therefore, if the sampling data at the time t (n) is in the buffer memory 70, by reading it out, it is possible to obtain the received light amount data at the equally spaced position P = D (1-an). Since data sampled at fixed time intervals is stored in the buffer memory in the order of addresses, if the time t (n) is obtained, the corresponding address is obtained, and desired light receiving amount data is obtained. Actually, the address corresponding to the time t (n) calculated does not become an integer, but a fraction below the decimal point occurs. In this case, processing for rounding to an integer value is performed, or interpolation processing is performed using sampling data of two adjacent addresses. In the present embodiment, the accuracy is increased by performing the interpolation processing. Details will be described later. Note that the order of addresses to be read out needs to be reversed for sampling data obtained by reverse scanning when performing reciprocal scanning.
[0056]
Next, the above description will be supplemented with specific numerical values. For example, when the scanning cycle T is 125 μsec (oscillation frequency 8 kHz) and the sampling frequency is 30 MHz, 3750 sampling data can be obtained per scanning cycle. This is the case of reciprocal scanning, and 1875 pieces of sampling data are obtained per one-directional scanning.
[0057]
As described above, 3750 pieces of sampling data of the reciprocating scanning are obtained and stored in the buffer memory 70 in the order of addresses. Consider a case where a part of the stored data is read out at a rate of 18 MHz and displayed on a screen. At this time, the number of data read out during the horizontal scanning cycle of 125 μsec is 2250, which is 1125 per one-direction scanning.
[0058]
When the normalized position signal changes from +1 (left end) to -1 (right end), it means that the screen has been scanned from the left end to the right end. Assuming that the position data corresponding to the n-th position is 1-2n / 1125, the time t (n) at this time is represented by t (n) = (T / (2π)) arccos (1-2n / 1125). You.
[0059]
Since the data position (corresponding to an address) at time T / 2 is 1875, the data position m at time t (n) is m = 1875 × tn × 2 / T. If a value obtained by rounding the obtained data position m to an integer is set as the address m again, the data at the address n is obtained by reading the data at the address m at the n-th read pulse.
[0060]
In practice, the calculated data position m includes a fraction after the decimal point. For example, when m = 123.4 is obtained, if interpolation processing is performed using two data of addresses m = 123 and m = 124, highly accurate data at the actual data position m = 123.4 is obtained. Can be Assuming that the data at address m = 123 is d1 and the data at address m = 124 is d2, the data d at data position m = 123.4 is d = 0.6d1 + 0.4d2 by linear interpolation. In general, if the fraction below the decimal point (corresponding to the interpolation coefficient) of the data position m is q, linear interpolation calculation using data d1 of an address of an integer part of m and data d2 of an adjacent (+1) address is performed. Thus, d = (1−q) d1 + q × d2.
[0061]
When the relationship between the equally-spaced position and the address of the buffer memory corresponding thereto is determined at the stage when the measurement conditions such as the scanning mode are set and stored as an address correspondence table, the above-mentioned fraction (interpolation coefficient ) May be stored in association with the equally spaced positions. FIG. 8 is a diagram illustrating an example of an address correspondence table indicating the correspondence between the equally-spaced position n, the read address m, and the interpolation coefficient q. The interpolation calculation unit 74 in the block diagram shown in FIG. 6 executes the above-described linear interpolation processing using the interpolation coefficients stored in the address correspondence table.
[0062]
FIG. 9 is a block diagram illustrating a configuration example of the interpolation calculation unit 74. The interpolation operation unit 74 includes a main operation circuit 81, a +1 addition circuit 82, and a one's complement operation circuit 83. The address m obtained from the address correspondence table 73 becomes +1 through the +1 adding circuit 82 and is given to the address port of the first buffer memory 70A, and the address m is given to the address port of the second buffer memory 70B as it is. The interpolation coefficient q obtained from the address correspondence table 73 becomes 1-q via the one's complement operation circuit 83, and q and 1-q are given to the main operation circuit 81.
[0063]
The main operation circuit 81 multiplies the value (1-q) d1 obtained by multiplying the data d1 read from the first buffer memory 70A by 1-q and the data d2 read from the second buffer memory 70B by q. By adding the value q × d2, data d = (1−q) d1 + q × d2 at the data position (m + q) is calculated. The data for each position at equal intervals calculated in this way is output from the interpolation calculation unit 74.
[0064]
In FIG. 9, first and second buffer memories 70A and 70B are provided for simultaneously reading out the data d1 of the address m and the data d2 of the address m + 1 and performing a high-speed operation, and the sampling data from the first AD converter 41 is provided. Are stored in the first and second buffer memories 70A and 70B. When the data d1 at the address m and the data d2 at the address m + 1 are read out with a time lag, one buffer memory 70 is sufficient as shown in FIG.
[0065]
In the description so far, as shown in FIG. 7, all the sampling data from the left end position (+ D) to the right end position (-D) of the scanning range is to be read. Is controlled by using a gate signal so as not to use. That is, in FIG. 7, the sampling data within a predetermined range centered on the center of the position (zero cross) is read.
[0066]
FIG. 10 is a diagram illustrating an example of read control using a gate signal. (A) schematically shows scanning after converting sampling data into data for each position at equal intervals using an address correspondence table. Also, a state is shown in which the process of reversing the address order for sampling data obtained on the return path (reverse scan) in reciprocal scanning has been completed. (B) schematically shows a data string for each position at equal intervals. (C) represents the gate signal described above. Data reading is valid only during the period when this signal is at the H level.
[0067]
FIG. 10D shows a state where the gate signal is slightly shifted to the front side of the time axis. Thus, by shifting the gate signal, the center of the data range to be read can be shifted from the center of the scanning range. This enables the offset adjustment described above. Of course, it is also possible to adjust the offset by shifting the value of the address (data position) m with respect to the equally spaced position n in the address correspondence table without shifting the gate signal.
[0068]
Further, the value of the address (data position) m with respect to the equally spaced position n in the address correspondence table is corrected according to the delay time of the signal processing system. In the case of one-way scanning, this delay time correction is not necessarily required, but in the case of reciprocal scanning, the delay time acts in the opposite directions on the outward and return paths, so that the delay time correction is required.
[0069]
FIG. 10E shows a gate signal when performing one-way scanning. In this way, by changing the gate signal, it is possible to make only the reading of data in one direction (outgoing path) effective.
[0070]
FIG. 11 is a flowchart of a process of generating an address correspondence table, which is executed when setting of measurement conditions such as a scanning mode is completed. In step # 101, a data position m is calculated by an inverse trigonometric function (arccos) operation. In step # 102, the correction amount of the delay time of the signal processing system is mainly added. In the next step # 103, the offset adjustment amount is added.
[0071]
In the next step # 104, the integer part of the data position m obtained as described above is set as the address m again, and the decimal part is set as the interpolation coefficient q. Thus, an address correspondence table including the equally spaced positions n, the addresses m corresponding to the respective positions n, and the interpolation coefficients q is generated.
[0072]
At the next step # 105, it is checked whether or not reciprocal scanning has been set. If reciprocal scanning has been set, a gate signal for reciprocal scanning is selected (step # 106). If the reciprocal scanning is not set, the gate signal for one-way scanning as described above is selected (step # 107). In the next step # 108, the address correspondence table generated as described above is stored, and the process ends.
[0073]
As described above, the embodiments of the present invention have been described while appropriately including the modified examples. However, the present invention is not limited to the above embodiments, and can be implemented in various forms. For example, the confocal microscope of the above embodiment is a reflection type microscope, but the present invention can also be applied to a transmission type confocal microscope. In the case of a transmission microscope, laser light of a confocal optical system and white light of a non-confocal optical system are emitted from the back surface of the sample. The light source of the confocal optical system may include not only a monochromatic light source including a laser light source but also a light source including a plurality of wavelengths. The light source of the non-confocal optical system can be replaced by natural light or room light.
[0074]
【The invention's effect】
As described above, according to the confocal microscope of the present invention, non-equidistant sampled data sampled at regular intervals by the resonance scanner is used to convert the equally-spaced pixel data by the address conversion process using the buffer memory. It can be converted into light receiving information of the position. Thus, it is possible to eliminate the distortion in which the central portion of the generated image is extended and the peripheral portion is blocked. In addition, by making the order of the address for reading out the sampling data in the forward scanning and the order of the address for reading out the sampling data in the reverse scanning opposite to each other, it is possible to relatively easily realize the reciprocating scanning by the resonance type scanner. it can.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a confocal microscope system according to an embodiment of the present invention.
FIG. 2 is a graph illustrating an example of received light amount data that changes according to the Z-direction position of an objective lens.
FIG. 3 is a block diagram illustrating a hardware configuration example in which an external computer system is connected to a controller of the confocal microscope system.
FIG. 4 is a diagram showing an example of a screen display for setting a measurement condition including selection of a scanning mode.
FIG. 5 is an enlarged view of an area for setting measurement conditions in the screen display of FIG. 4;
FIG. 6 is a block diagram showing a configuration for obtaining light reception amount data at equal intervals from sampling data of the light reception amount obtained by scanning using a resonance type scanner.
FIG. 7 is a graph showing a change in the position of a scanning spot by a resonance-type scanner (more precisely, a change in the angle of a mirror).
FIG. 8 is a diagram illustrating an example of an address correspondence table indicating a correspondence relationship between an equally-spaced position n, a read address m, and an interpolation coefficient q.
FIG. 9 is a block diagram illustrating a configuration example of an interpolation operation unit.
FIG. 10 is a diagram illustrating an example of read control using a gate signal.
FIG. 11 is a flowchart of an address correspondence table generation process that is executed when setting of measurement conditions such as a scanning mode is completed.
[Explanation of symbols]
1 Confocal microscope system
2 Confocal optical system
14a Horizontal scanning device (resonant scanner)
14b Vertical scanning device (galvano scanner)
19 Light receiving element
47,51 display device
70, 70A, 70B buffer memory
73 Address Correspondence Table
74 interpolation calculator

Claims (3)

A sample surface within a measurement range determined by an optical magnification is scanned with light, light from the sample is received by a light receiving element via a confocal optical system, and height distribution information or an image of the sample surface based on the received light information. A confocal microscope for acquiring information,
At least a part of the light scanning means for scanning the sample surface with light is configured using a resonance type scanner,
The light receiving information obtained by receiving the light from the sample scanned with light by the resonance scanner with the light receiving element is sampled at regular intervals and stored in a buffer memory,
By reading out the sampling data stored in the buffer memory at non-equidistant addresses in accordance with the address correspondence table obtained by the operation processing including the inverse trigonometric function operation, pixels at substantially equal intervals on the sample surface within the measurement range are read out. A confocal microscope characterized by obtaining light receiving information for each position. In order to realize the reciprocating scanning by the resonance type scanner, in the address correspondence table, the order of the address for reading the sampling data in the forward scanning and the order of the address for reading the sampling data in the reverse scanning are set to be opposite to each other. The confocal microscope according to claim 1, wherein: When reading sampling data from the buffer memory at non-equidistant addresses in accordance with the address correspondence table, an interpolation process is performed using sampling data at two adjacent addresses to thereby obtain light reception information at the pixel positions at substantially equal intervals. 3. The confocal microscope according to claim 1, wherein the accuracy of the confocal microscope is increased.

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