JP4303465B2 - Confocal microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
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 determines the height of the sample surface based on the received light information. More specifically, the present invention relates to a confocal microscope in which at least a part of an optical scanning unit is configured by a resonance scanner.
[0002]
[Prior art]
In measuring 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 the light receiving element via the confocal optical system, and based on the amount of received light, information such as the height distribution information of the sample and an ultra-deep image (an image with a very deep focal depth) Image information is acquired. 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 of light entering the light receiving element through the pinhole of the confocal optical system (the amount of received light) changes. The amount of light received is maximized when the surface of the camera is in focus. Therefore, the 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]
In addition, 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 focal depth can be obtained. Obtainable. This image is a so-called ultra-deep image. Alternatively, if the distance between the sample and the objective lens when the maximum amount of received light is obtained at any pixel of interest is fixed, the amount of light received by the pixel of the pixel of interest and the portion where the height difference is large is significantly reduced. A bright image (so-called slice image) is obtained only in a portion having the same height as the pixel.
[0004]
The optical scanning unit of the confocal microscope as described above is usually a two-dimensional optical scanning unit including a horizontal scanning unit and a vertical scanning unit. 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]
The galvano scanner can change the angle of the mirror at a substantially constant speed, whereas the resonant (resonant type) scanner changes the angle of the mirror by vibration using a sine wave as the drive signal, so the angular speed is sinusoidal. It becomes maximum near the center of the scanning range, and the angular velocity approaches zero as it approaches both ends of the scanning range. Therefore, a dimensional difference in the scanning direction per pixel between the center and both ends of the image obtained by optical scanning appears as image distortion. That is, distortion occurs in which the central portion of the image extends and the peripheral portion is clogged.
[0006]
In order to suppress this image distortion, it is necessary to use a portion in the scanning range of the resonant scanner near the center where the angular velocity changes relatively linearly, that is, a portion that changes substantially linearly around the zero cross of the sine wave. There is. However, as in the case of performing offset adjustment for alignment with a color image obtained by a color image sensor, for example, it is necessary to secure a certain adjustment region outside the region that is actually used. For this reason, it is advantageous that the widest possible region including the portion with nonlinear distortion near both ends in the scanning range of the resonant scanner can be effectively used.
[0007]
Further, due to the characteristics of the resonant scanner, the required time for the forward path and the backward path is the same. Therefore, when only the forward path is used for scanning, only half of one cycle is used, and the remaining half is wasted time. Therefore, it is conceivable to perform reciprocating scanning that also makes effective use of the return path.
[0008]
As described above, as a conventional technique for effectively utilizing the portion with nonlinear distortion near both ends in the scanning range of the resonant scanner, the back surface of the mirror of the resonant scanner is irradiated with auxiliary laser light, There is a method in which a sample is sampled at non-equal intervals in time obtained by scanning an equally spaced pattern with reflected light (see, for example, JP-A-2000-147395). At this time, reciprocal scanning becomes possible by changing the order of the sampling data in the return path using the line memory.
[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 scanner with auxiliary laser light and scanning the equidistant pattern with the reflected light, and the structure is complicated accordingly. become. In addition, precision processing technology is also required.
[0010]
In view of the above-described problems, the present invention effectively utilizes a portion with nonlinear distortion near the both ends of the scanning range of the resonance scanner without adding a new optical system, and performs reciprocal scanning. An object of the present invention is to provide a confocal microscope that is 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 through a confocal optical system, and the sample is based on the light reception information. A confocal microscope for acquiring surface height distribution information or image information, wherein at least a part of an optical scanning means for scanning the surface of a sample with light is configured using a resonance scanner, and light is emitted by the resonance scanner. The received light information obtained by receiving the light from the sample scanned with the light receiving element is sampled at regular intervals. Integer type memory address order The sampling data stored in the buffer memory is stored at non-uniform intervals according to the address correspondence table obtained by the arithmetic processing including the inverse trigonometric function calculation. Integer type memory address Specify from the buffer memory By reading, light reception information for each pixel position at substantially equal intervals on the sample surface within the measurement range is obtained.
[0012]
According to such a configuration, it is not a method of adding an optical system as described in the prior art, but non-uniformly sampled data sampled at regular intervals by an address conversion process using a buffer memory. Can be converted into light reception information at the pixel position. As a result, it is possible to eliminate the distortion in which the central portion of the generated image extends and the peripheral portion is clogged.
[0013]
In a preferred embodiment, in order to realize reciprocal scanning by a resonance scanner, in the address correspondence table, the order of addresses from which sampling data is read in forward scanning and the order of addresses from which sampling data is read in backward scanning are opposite to each other. I have to.
[0014]
In another preferred embodiment, when sampling data is read from the buffer memory at non-uniformly spaced addresses in accordance with the address correspondence table, interpolation processing is performed using the sampling data of two adjacent addresses, thereby approximately equally spaced pixels. Increase the accuracy of light reception information of position. That is, since the addresses corresponding to the equally spaced pixel positions obtained by the inverse trigonometric function calculation are values including fractions after the decimal point, interpolation processing is performed using the sampling data of two adjacent addresses with this fraction as an interpolation coefficient. Doing so increases the accuracy.
[0015]
DETAILED DESCRIPTION OF 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 drive circuit 44 for the confocal microscope, a first AD converter 41, a CCD drive circuit 43, and a second AD converter. 42, an objective lens moving mechanism 40, a controller including a control unit 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 a sample wk with monochromatic light (for example, laser light), a first collimating lens 11, a polarizing beam splitter 12, a quarter wavelength plate 13, a horizontal scanning device 14a, and vertical scanning. A device 14b, 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 are included.
[0018]
As the light source 10, for example, a semiconductor laser that emits blue laser light is used. The laser light emitted from the light source 10 driven by the laser driving circuit 44 passes through the first collimating lens 11, the optical path is bent by the polarization beam splitter 12, and passes through the quarter wavelength plate 13. Thereafter, after being deflected in the horizontal (lateral) direction and the vertical (longitudinal) direction by the horizontal scanning device 14 a and the vertical scanning device 14 b, it passes through the first relay lens 15 and the second relay lens 16 and is sampled by the objective lens 17. The light is condensed on the surface of the sample wk placed on the stage 30.
[0019]
The horizontal scanning device 14a is constituted by a resonant (resonance type) scanner, and the vertical scanning device 14b is constituted by a galvano (electromagnetic type) scanner. The surface of the sample wk is scanned with the laser beam by deflecting the laser beam in both the horizontal and vertical directions. For convenience of explanation, the horizontal direction is referred to as the X direction, and the vertical direction is referred to as the 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 of the focus of the objective lens 17 and the sample wk can be changed.
[0020]
However, the distance between the focal point of the objective lens 17 and the sample wk in the optical axis direction can be changed by other methods. 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 this 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 has a stage manual operation mechanism. It can be displaced in X direction, Y direction and Z direction by manual operation via 31. Further, 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, operation of an up / down key).
[0022]
The laser beam reflected by the sample wk follows the above optical path in reverse. That is, it passes through the objective lens 17, the second relay lens 16, and the first relay lens 15, and again passes through the quarter wavelength plate 13 through the horizontal scanning device 14a and the vertical scanning device 14b. As a result, the laser beam passes through the polarization beam splitter 12 and is collected 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. An electrical signal corresponding to the amount of received light is supplied to the first AD converter 41 via an output amplifier and a gain control circuit (not shown), and converted into a digital value.
[0023]
With the confocal optical system 2 configured as described above, height (depth) information of the sample wk can be acquired. 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 between the focal point of the objective lens 17 and the sample wk in the optical axis direction changes. Then, 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 through the above optical path, and almost all the laser light is collected. 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. On the contrary, in a state where the focus of the objective lens 17 is deviated from the surface of the sample wk, the laser light collected by the imaging lens 18 is focused at a position deviated from the pinhole plate 9, so that some lasers 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 received light amount of the light receiving element 19 is detected while driving the objective lens 17 in the Z direction (optical axis direction) at any point on the surface of the sample wk, the objective lens 17 when the received light amount becomes maximum. The position in the Z direction (distance between the focal point of the objective lens 17 and the sample wk in the optical axis direction) can be uniquely determined as height information.
[0026]
Actually, each time the objective lens 17 is moved by one step (one pitch), the surface of the sample wk is scanned by the horizontal scanning device 14a and the vertical scanning device 14b 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, received light amount data that changes in accordance with the position in the Z direction 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 in accordance with the position of the objective lens 17 in the Z direction. Based on such received light amount data, the maximum received light amount and the position in the Z direction 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, it can be displayed as a two-dimensional distribution of brightness by converting the height data into luminance data. By converting the height data into color difference data, the height distribution can also be displayed as a color distribution.
[0029]
Further, a surface image (black and white image) of the sample wk is obtained from a luminance signal having the received light amount obtained for each point (pixel) in the XY scanning range as luminance data. If a luminance signal is generated by using the maximum amount of received light in each pixel as luminance data, an ultra-deep image having a very deep focal depth in focus at each point having a different surface height can be obtained. In addition, if the height (Z-direction position) at which the maximum received light amount is obtained at any target pixel is fixed, the received light amount of the pixel of the target pixel portion and the portion where the height difference is large is significantly reduced. A bright image is obtained only at the same height.
[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 photographing a color image). A CCD (image sensor) 24 and the like are included. Further, 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 coincide.
[0031]
For example, a white lamp is used as the white light source 20, but natural light or room light may be used. 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 condensed 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, and enters the color CCD 24 to form an image. The color CCD 24 is provided at a position conjugate to or close to the conjugate with 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 the 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 synthesized, and the color super-depth with a deep focus depth that is in focus at all pixels. Images can also be generated and displayed.
[0034]
The controller including the control unit 46 also controls the processing related to the color image as described above. An input device 48 such as a console (console console) and a display device 47 such as a CRT (cathode ray tube) or 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 in accordance with the guidance displayed on the screen of the display device 47. For example, the Z direction movement range (measurement range) and movement pitch of the objective lens 17 are set. Alternatively, by setting the light receiving sensitivity (PMT gain) of the light receiving element 19 and the attenuation amount by the ND filter in accordance with the light reflectance of the surface of the sample wk, the ultra-deep image and slice image displayed on the screen are appropriate. Adjust the brightness (brightness). The shutter speed, gain, and white balance for obtaining a color image by the color CCD 24 are set. In addition, various scanning modes as described later are set.
[0036]
Further, the confocal microscope system 1 (controller) of the present embodiment is also provided with an interface for connecting an external computer system such as a personal computer. By connecting an external computer system in which dedicated software is installed to the confocal microscope system 1, the measurement conditions as described above are set on the screen of the external computer system, or the image acquired by the confocal microscope system 1 is processed. Can be displayed on the screen of an external computer system and processed.
[0037]
FIG. 3 is a block diagram illustrating a hardware configuration example in which the external computer system 50 is connected to the 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 (may be another pointing device) 53, an RS232C, a USB (universal serial bus), a communication interface 54 such as IEEE1394, and a processing device (CPU). 55, a main memory 56 that is a semiconductor storage medium, a fixed disk device 57 that is an auxiliary storage device, and a removable disk device 58.
[0038]
Dedicated software for controlling the confocal microscope system 1 is supplied in a state of being stored in a storage medium 59 such as a CD-ROM, and is removed from the storage medium 59 by a removable disk device 58 such as a CD-ROM drive device. 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 the measurement conditions of the confocal microscope system 1 and processing of the image obtained as a result of the measurement. Next, selection of the scanning mode in the screen display for setting 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 the screen display 60 displayed on the display device 51, the left region 61 is obtained from the color image obtained from the color CCD 24 of the non-confocal optical system 3 of the confocal microscope system 1 or the light receiving element 19 of the confocal optical system 2. This is a region for displaying the confocal image or the height distribution measurement result, and a vertically long region 62 for setting measurement conditions is displayed on the right side.
[0041]
FIG. 5 is an enlarged view of a region 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 place where “scan setting” is displayed. When the downward triangular mark at 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 of “normal”, “part 1/2”, “part 1/3”, “skip”, and “skip 1/2” as options. One scan 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 in the XY plane determined by the optical magnification, and the maximum number of scanning lines (for example, 768). In “Part 1/2”, the vertical scanning range is ½ of the entire central portion, and the number of scanning lines is ½ (for example, 384 lines) accordingly. In “Part 1/3”, the vertical scanning range is 1/3 of the entire center, and the number of scanning lines is also 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 “skip 1/2”, the interval between adjacent scanning lines is doubled, and the vertical scanning range is 1/2 of the entire central portion. Therefore, the number of scanning lines becomes 1/4. In any scanning mode, the horizontal scanning range does not change and is equal to the horizontal length of the entire measurement range. Further, when the interval between adjacent scanning lines is doubled by skipping, pixel data between the scanning lines is generated by interpolation processing.
[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 thereof. When the user checks the check box 65 using the mouse 53, the reciprocating scan is set for the horizontal scan, and when the check is removed, the normal one-way scan is set.
[0045]
As described above, the horizontal scanning device 14a for horizontal scanning is constituted by a resonant (resonant) scanner, and the reflection surface of the mirror vibrates at a predetermined frequency in a self-excited manner by a sine wave drive signal. Accordingly, since the required time for the forward path and the backward path are the same (half cycle) and the phase change is the same, reciprocal scanning is possible. When reciprocating scanning is performed, compared with the case of unidirectional scanning, if the number of horizontal scanning per vertical scanning is the same, the apparent number of scanning lines can be doubled, 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 there is almost no decrease in measurement accuracy. If the number of horizontal scans per vertical scan is reduced to half, the time required for the entire measurement is reduced to nearly half.
[0046]
However, since the resonance 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 approaches the 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 the image obtained by optical scanning appears as image distortion. That is, distortion occurs in which the central portion of the image extends and the peripheral portion is clogged.
[0047]
As a method of eliminating this image distortion, in the confocal microscope system 1 of the present embodiment, the received light information is sampled at regular intervals, stored in the buffer memory, and the address correspondence obtained by the arithmetic processing including the inverse trigonometric function calculation is performed. According to the table, the sampling data stored in the buffer memory is read out at non-uniformly spaced addresses, thereby obtaining the light receiving information of the pixel positions at substantially regular intervals 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 equidistant positions from received light amount sampling data 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 regular intervals according to the 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 generation unit 72 performs addressing of the buffer memory 70 in accordance with an address correspondence table 73 described later, whereby (a part of) the stored data in the buffer memory 70 is read at unequal intervals. As a result, the received light amount data for each equally spaced pixel position is read out. As will be described later, the address correspondence table 73 is obtained by arithmetic processing including inverse trigonometric function calculation at the stage where measurement conditions including the scanning mode are set, and is stored in the memory.
[0050]
The data read from the buffer memory 70 at non-uniformly spaced addresses may be used as received light amount data for each equally spaced pixel position, but in the configuration of FIG. Thus, the accuracy of the received light amount data for each equally spaced pixel position is increased. That is, since the addresses corresponding to the equally spaced pixel positions obtained by the inverse trigonometric function calculation are values including fractions after the decimal point, interpolation processing is performed using the sampling data of two adjacent addresses with this fraction as an interpolation coefficient. Doing so increases the accuracy. Details of this interpolation calculation will be described later.
[0051]
When the reciprocal scanning mode is selected when generating the address correspondence table 73, the order of addresses from which sampling data is read in forward scanning and the order of addresses from which sampling data is read in backward scanning are opposite to each other. I have to.
[0052]
Next, the address correspondence table 73 will be described. FIG. 7 is a graph showing changes in the position of the scanning spot (light spot) by the resonant scanner (more precisely, the change in the angle of the mirror). For example, it is assumed that the position of the light spot changes from the left end (+ D) to the right end (−D) when the time changes to t0, t1,. Of course, the actual sampling time is finer.
[0053]
Since the angle of the mirror changes in the resonance scanner in a sine wave shape, the position of the scanning spot corresponding to the equally spaced times t0, t1,... T14 is the center of the scanning range as shown in FIG. It becomes a non-equal interval that extends between and clogs at both ends. Therefore, when the received light amount data sampled at regular intervals is used as it is as pixel data at equal intervals, distortion occurs in which the central portion of the image extends and the peripheral portion is clogged. FIG. 7B shows a state in which the times t0, t1,... T14 are non-uniformly spaced when the positions of the scanning spots are equally spaced.
[0054]
7A and 7B, when the position of the scanning spot is P, the scanning cycle is T, and the times t0, t1,..., T14 are represented by the variable t, the trigonometric function (cosine function) cos is 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 the moving pitch (corresponding to the pixel pitch) of the position of the scanning spot is a and n is a natural number, the time t (n) corresponding to the position of P = D (1-an) is t (n ) = (T / (2π)) arccos (1-an).
[0055]
Therefore, if the sampling data at time t (n) is stored in the buffer memory 70, the received light amount data at the equally spaced positions P = D (1-an) can be obtained by reading it out. Since the buffer memory stores data sampled at regular time intervals in the order of addresses, when the time t (n) is obtained, the corresponding address is obtained and desired received light amount data is obtained. Actually, the address corresponding to the time t (n) obtained by calculation is not an integer, and a fractional part occurs. In this case, processing to round 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 improved by performing an interpolation process. Details will be described later. It should be noted that for the sampling data obtained by reverse scanning when performing reciprocal scanning, it is necessary to reverse the order of addresses to be read.
[0056]
Next, the above description will be supplemented with specific numerical values. For example, when the scanning cycle T = 125 μsec (oscillation frequency 8 kHz) and the sampling frequency 30 MHz, 3750 pieces of sampling data can be obtained per scanning cycle. This is a case of reciprocal scanning, and 1875 sampling data are obtained per one-way scanning.
[0057]
As described above, 3750 pieces of sampling data for reciprocal 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 the screen. At this time, the number of data read out during the horizontal scanning period of 125 μsec is 2250, which is 1125 per one-way scanning.
[0058]
When the normalized position signal changes from +1 (left end) to -1 (right end), scanning is performed from the left end to the right end of the screen. 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). The
[0059]
Since the data position (corresponding to the address) at time T / 2 is 1875, the data position m at time t (n) is m = 1875 × tn × 2 / T. Assuming that the value obtained by rounding the obtained data position m to an integer is the address m, the data at the address n can be obtained by reading the data at the address m at the nth read pulse.
[0060]
Actually, the data position m obtained by calculation includes a fractional part. For example, when m = 123.4 is obtained, high-precision data at the actual data position m = 13.4 can be obtained by performing interpolation using two data of addresses m = 123 and m = 124. It is done. If the data at the address m = 123 is d1, and the data at the address m = 124 is d2, the data d at the data position m = 13.4 is d = 0.6d1 + 0.4d2 by linear interpolation. In general, if q is the fractional part of the data position m (corresponding to the interpolation coefficient), the linear interpolation calculation using the data d1 at the address of the integer part of m and the data d2 at the adjacent (+1) address. Thus, d = (1−q) d1 + q × d2.
[0061]
When the relationship between the equidistant position and the corresponding buffer memory address is obtained and stored as an address correspondence table at the stage when the measurement conditions such as the scanning mode are set, the above-mentioned fraction (interpolation coefficient) together with the integer value as the address is stored. ) May be stored in correspondence with the equally spaced positions. FIG. 8 is a diagram showing an example of an address correspondence table showing the correspondence between the equidistant 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 linear interpolation process as described above using the interpolation coefficient 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 calculation unit 74 includes a main calculation circuit 81, a +1 addition circuit 82, and a one's complement calculation circuit 83. The address m obtained from the address correspondence table 73 becomes m + 1 through the +1 addition 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. Further, the interpolation coefficient q obtained from the address correspondence table 73 becomes 1-q through the one's complement arithmetic circuit 83, and q and 1-q are given to the main arithmetic circuit 81.
[0063]
The main arithmetic circuit 81 multiplies the data d1 read from the first buffer memory 70A by 1-q (1-q) d1 and the data d2 read from the second buffer memory 70B by q. The value q × d2 is added to calculate data d = (1−q) d1 + q × d2 at the data position (m + q). Data for each equally spaced position 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 to simultaneously read out data d1 at address m and data d2 at address m + 1 and perform high-speed computation. 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 while being shifted in time, one buffer memory 70 is sufficient as shown in FIG.
[0065]
In the description so far, as shown in FIG. 7, the sampling data from the left end position (+ D) to the right end position (−D) of the scanning range are all read out. The data read control is performed using the gate signal so that it is not used. That is, in FIG. 7, sampling data within a predetermined range centering on the central portion (zero cross) of the position is read.
[0066]
FIG. 10 is a diagram illustrating an example of read control using a gate signal. (A) schematically shows scanning after the sampling data is converted into data for each equally spaced position using the address correspondence table. Further, a state is shown in which the processing for reversing the address order is performed on the sampling data obtained in the return path (reverse scan) in the reciprocating scan. (B) has shown typically the data row | line for every position of 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 in which 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. As a result, the above-described offset adjustment becomes possible. Of course, the offset can be adjusted 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 unidirectional scanning, this delay time correction is not necessarily required. However, in the case of reciprocating scanning, the delay time acts in the opposite direction on the forward path and the backward path, so that the delay time correction is necessary.
[0069]
FIG. 10E shows a gate signal when unidirectional scanning is performed. In this way, by changing the gate signal, only one-way (outward) data reading can be made effective.
[0070]
FIG. 11 is a flowchart of the address correspondence table generation process executed when the setting of the measurement conditions such as the scanning mode is completed. In step # 101, the data position m is calculated by inverse trigonometric function (arccos) calculation. In step # 102, the correction amount of the delay time of the signal processing system is mainly added. In 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, and the decimal part is set as the interpolation coefficient q. In this way, an address correspondence table is generated which includes positions n at equal intervals, addresses m and interpolation coefficients q corresponding to the respective positions n.
[0072]
In the next step # 105, it is checked whether or not reciprocating scanning is set. If reciprocating scanning is set, a gate signal for reciprocating scanning is selected (step # 106). If reciprocal scanning is not set, the gate signal for unidirectional 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 mentioned above, although embodiment of this invention was described including a modification suitably, this invention is not restricted to said embodiment, It can implement with a various form. 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 type microscope, laser light from a confocal optical system and white light from a non-confocal optical system are irradiated 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 plurality of wavelengths. The light source of the non-confocal optical system can be replaced with natural light or room light.
[0074]
【The invention's effect】
As described above, according to the confocal microscope of the present invention, non-equal-interval sampling data sampled at regular intervals by the resonant scanner is converted into equal-interval pixels by address conversion processing using a buffer memory. It can be converted into light reception information of the position. As a result, it is possible to eliminate the distortion in which the central portion of the generated image extends and the peripheral portion is clogged. In addition, by making the order of addresses for reading sampling data in forward scanning and the order of addresses for reading sampling data in backward scanning opposite to each other, it is possible to realize reciprocating scanning with a resonance scanner relatively easily. 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 showing an example of received light amount data that changes in accordance with the position of the objective lens in the Z direction.
FIG. 3 is a block diagram illustrating a hardware configuration example in which an external computer system is connected to a controller of a confocal microscope system.
FIG. 4 is a diagram showing an example of a screen display for setting measurement conditions including selection of a scanning mode.
FIG. 5 is an enlarged view of a region for setting measurement conditions in the screen display of FIG. 4;
FIG. 6 is a block diagram showing a configuration for obtaining received light amount data at equally spaced positions from received light amount sampling data obtained by scanning using a resonance type scanner.
FIG. 7 is a graph showing a change in the position of a scanning spot (more precisely, a change in the angle of a mirror) by a resonant scanner.
FIG. 8 is a diagram showing an example of an address correspondence table showing a correspondence relationship between an equidistant position n, a read address m, and an interpolation coefficient q.
FIG. 9 is a block diagram illustrating a configuration example of an interpolation calculation 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 to be executed when measurement conditions such as a scanning mode have been set.
[Explanation of symbols]
1 Confocal microscope system
2 Confocal optics
14a Horizontal scanning device (resonance type 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 (2)

The sample surface within the measurement range determined by the optical magnification is scanned with light, the light from the sample is received by the light receiving element via the confocal optical system, and the height distribution information or image of the sample surface based on the received light information A confocal microscope for acquiring information,
At least a part of the optical scanning means for scanning the sample surface with light is configured using a resonant scanner,
Light reception information obtained by receiving light from the sample scanned with light by the resonance type scanner with the light receiving element is sampled at regular intervals and stored in the buffer memory in the order of addresses of integer type memory addresses ,
According to the address correspondence table obtained by the arithmetic processing including the inverse trigonometric function calculation, when reading the sampling data accumulated in the buffer memory from the buffer memory by designating the integer type memory addresses at non-uniform intervals, Interpolation processing is performed using sampling data of two adjacent memory addresses out of the integer type memory addresses, thereby obtaining light reception information for each substantially equidistant pixel position on the sample surface within the measurement range. Confocal microscope. In order to realize reciprocal scanning by the resonance type scanner, in the address correspondence table, the order of the integer type memory address from which the sampling data in the forward scanning is read out and the integer type memory address from which the sampling data in the backward scanning is read out The confocal microscope according to claim 1, wherein the first and second orders are in opposite directions.

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