JP2010262104A - Scanning confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To reliably observe a sample whose luminance difference in a displayed image is large with one image regarding a scanning confocal microscope. <P>SOLUTION: The scanning confocal microscope includes: a light source which outputs first light and second light having higher intensity than the first light; a scanning means which emit the first light for scanning from the light source onto the sample on the outgoing route and the second light onto the sample on the return route; a light detection means which detects light from the sample by scanning; and an image processing means which obtains an image made of a plurality of pixels based on the light detected by the light detection means and processes the obtained image. The image processing means includes: a determining means for determining whether or not the pixels of the second image obtained by emission of the second light onto the sample are pixels of a predetermined luminance or higher; and a correction means which corrects the luminance values of the pixels of the predetermined luminance or higher of the second image based on the luminance values of the corresponding pixels of the first image obtained by emission of the first light onto the sample. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a scanning confocal microscope.

  A scanning confocal microscope illuminates a sample in a point-like manner, for example, collects fluorescence from the sample on the confocal stop, and then detects the intensity of light passing through the confocal stop with a photodetector. The surface information is acquired. Further, in the scanning confocal microscope, the surface information of a wide range of the sample can be acquired by scanning the sample surface with the point illumination by various methods.

  In such a scanning confocal microscope, when the difference in luminance in the displayed image is large, that is, when a sample having a large difference between a bright part and a dark part is being observed, the image is not saturated on the image. Thus, it is necessary to adjust the detection sensitivity. In that case, adjustments must be made based on the bright area. If this happens, the data in the dark area will contain a lot of noise, so in general, the bright area will be compressed using a logarithmic amplifier (γ characteristic). In addition, the dark portion is brightened to increase the dynamic range of light detection, and the overall contrast is adjusted to be lowered. In addition, a technique using an LUT (Look Up Table) is disclosed in order to improve the visibility of dark parts.

  However, when observing a sample with a large luminance difference in the displayed image, the use of a logarithmic amplifier increases the gain of the dark area, so that even if it is bright, circuit noise is greatly added. , S / N deteriorates from the start. The use of the LUT is merely adjusting the brightness of the appearance, and does not have an effect of improving the S / N determined at the time of A / D conversion. As a result, a high-quality image with little noise cannot be obtained. . In this case, it can be said that in this case, it is important to increase the light detection dynamic range without deteriorating the S / N of the light detection signal in the dark part of the sample.

JP 2006-87036 A

  On the other hand, when observing a sample with a large luminance difference in the displayed image, an image obtained by irradiating with relatively weak light and an image obtained by irradiating with relatively strong light Attempts have been made to observe a sample from two images. However, in this case, since it is necessary to observe the sample with two images, it takes a lot of time to establish the correspondence between the two images, and it is difficult to reliably observe the sample. There was a problem that there was.

  The present invention has been made to solve such a conventional problem, and provides a scanning confocal microscope capable of reliably observing a sample having a large luminance difference in a displayed image with one image. The purpose is to do.

  The scanning confocal microscope of the first invention includes a light source capable of outputting the first light and the second light having a higher intensity than the first light, and the first light from the light source in the forward path or Based on the scanning means for irradiating the sample with the second light in the return path to scan the sample, the light detecting means for detecting light from the sample by the scanning, and the light detected by the light detecting means Image processing means for acquiring an image composed of a plurality of pixels and performing image processing on the acquired image, wherein the image processing means is configured to output the second image acquired by irradiating the sample with the second light. A first image obtained by irradiating the sample of the first light with a determination unit for determining whether the pixel is a pixel having a predetermined luminance or higher; and a luminance value of the pixel having a predetermined luminance or higher in the second image. Based on the brightness value of the corresponding pixel of It characterized in that it has a correction means for.

  A scanning confocal microscope according to a second aspect of the present invention is the scanning confocal microscope according to the first aspect, wherein the predetermined luminance is a saturation luminance of the light detection means.

  A scanning confocal microscope according to a third aspect of the present invention is the scanning confocal microscope according to the first or second aspect of the invention, wherein the correcting means sets the second pixel value to the luminance value of the corresponding pixel of the first image. A luminance value of a pixel of the second image is corrected by multiplying a value obtained by dividing the intensity of the light by the intensity of the first light.

  A scanning confocal microscope according to a fourth invention is the scanning confocal microscope according to any one of the first to third inventions, wherein the scanning means reciprocally scans the same portion of the sample. .

  A scanning confocal microscope according to a fifth aspect of the invention is the scanning confocal microscope according to the fourth aspect of the invention, wherein the scanning means scans the sample with the first light in the forward path and the second light in the backward path. Scanning the sample.

  In the scanning confocal microscope of the present invention, a sample having a large luminance difference in the displayed image can be reliably observed with one image.

It is explanatory drawing which shows one Embodiment of the scanning confocal microscope of this invention. It is explanatory drawing which shows the relationship between the output of the photodetector in the scanning confocal microscope of FIG. 1, and a relative position. It is explanatory drawing which shows the control part of the scanning confocal microscope of FIG. It is explanatory drawing which shows operation | movement of the scanning confocal microscope of FIG. It is explanatory drawing explaining the reciprocating scan performed with the scanning confocal microscope of FIG. It is explanatory drawing explaining the reciprocating scan performed with a normal scanning confocal microscope. It is explanatory drawing which shows roughly the image of the sample obtained when it scanned with the light with weak laser power. It is explanatory drawing which shows the specific example of the image of FIG. It is explanatory drawing which shows roughly the image of the sample obtained when it scanned with the light with a strong laser power. It is explanatory drawing which shows the specific example of the image of FIG. It is explanatory drawing which shows the relationship between the intensity | strength of the light input into a photodetector, and an output value. It is explanatory drawing which shows the state which multiplied the luminance value of the 1st image by R times. It is explanatory drawing which shows the state which correct | amended the 2nd image. It is explanatory drawing which shows the brightness | luminance of the pixel of FIG. It is explanatory drawing which shows the specific example of the image of FIG.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 shows an embodiment of the scanning confocal microscope of the present invention.

  This scanning confocal microscope has an illumination optical system 11, a scanning optical system 13, an imaging optical system 15, a stage 16, a detection optical system 17, a control unit 19, and a monitor 21.

  The illumination optical system 11 includes a laser light source 23 and a reflecting mirror 25. The scanning optical system 13 has a two-dimensional scanning mechanism 27 and a scanner lens 29. The two-dimensional scanning mechanism 27 includes a first optical scanner 31 and a second optical scanner 33 (for example, a resonant scanner or a galvano scanner). The imaging optical system 15 includes a second objective lens 35 and a first objective lens 37. The stage 16 has a sample stage 38 on which the sample S is placed. The sample stage 38 can be finely moved in the vertical and horizontal directions. The detection optical system 17 includes a beam splitter 39, an imaging lens 41, a confocal stop 43, and a photodetector 45.

  In this scanning confocal microscope, the light emitted from the laser light source 23 passes through the beam splitter 39 and then enters the two-dimensional scanning mechanism 27. The two-dimensional scanning mechanism 27 scans the light beam two-dimensionally with the first optical scanner 31 and the second optical scanner 33 and guides the light to the scanner lens 29. The light beam from the scanner lens 29 is guided to the first objective lens 37 via the second objective lens 35. The light beam incident on the first objective lens 37 becomes a focused light and scans the surface of the sample S.

  The light reflected from the surface of the sample S is introduced from the first objective lens 37 into the beam splitter 39 via the two-dimensional scanning mechanism 27 and is reflected by the beam splitter 39. The reflected light is condensed on the pinhole 43 a of the confocal stop 43 by the imaging lens 41. The reflected light from other than the condensing point of the sample S is cut by the pinhole 43a, and only the light passing through the pinhole 43a is detected by the photodetector 45.

  In this scanning confocal microscope, the condensing position by the first objective lens 37 is optically conjugate with the pinhole 43 a of the confocal stop 43, and the sample S is at the condensing position by the first objective lens 37. In some cases, the reflected light from the sample S is collected on the pinhole 43a and passes through the pinhole 43a. When the sample S is at a position deviated from the condensing position by the first objective lens 37, the reflected light from the sample S is not condensed on the pinhole 43a and does not pass through the pinhole 43a.

In this scanning confocal microscope, as shown in FIG. 2, the output of the photodetector 45 is maximized when the sample S is at the condensing position Z ₀ of the first objective lens 37. Then, as the relative position between the first objective lens 37 and the sample S moves away from this position, the output of the photodetector 45 rapidly decreases. Due to this characteristic, if the condensing point is two-dimensionally scanned by the two-dimensional scanning mechanism 27 and the output of the light detector 45 is imaged in synchronism with the two-dimensional scanning mechanism 27, the portion of the sample S at a certain height is obtained. Only an image is obtained, and an image (confocal image) obtained by optically slicing the sample S is obtained.

  FIG. 3 is a block diagram illustrating a configuration of the control unit 19 of the above-described scanning confocal microscope.

  The control unit 19 includes a microscope control unit 47, an image processing unit 49, and a CPU 51.

  The microscope control unit 47 is connected to the laser light source 23, the two-dimensional scanning mechanism 27, the stage 16, and the like. Then, the laser light source 23, the two-dimensional scanning mechanism 27, the stage 16, and the like are controlled by an input such as start and stop of scanning from the input unit 53.

  The image processing unit 49 receives the signal photoelectrically converted by the light detector 45 and the timing signal from the two-dimensional scanning mechanism 27 and forms an image. Then, the surface information of the sample S can be obtained by displaying an image on the monitor 21.

  In this embodiment, a photodetector 45 having a function of amplifying an electrical signal at the electronic level, such as a photomultiplier tube, is employed as the photodetector 45. The reason why such a detector is used as the photodetector 45 is that high S / N amplification that cannot be achieved by electric circuit amplification can be performed. In addition, you may employ | adopt things other than a photomultiplier tube, for example, APD (avalanche photo diode), etc. as the photodetector 45. FIG.

  FIG. 4 is a flowchart showing the operation of the above-described scanning confocal microscope.

  Step S1: When a scanning start signal is input from the input unit 53, the CPU 51 of the control unit 19 first acquires image data of the sample S.

  Acquisition of image data is performed by scanning the sample S by scanning the two-dimensional scanning mechanism 27. FIG. 5 schematically shows the sample S, and a region P indicated by a small square is a region corresponding to one pixel of the image data. In this embodiment, the area of 1 line L is scanned back and forth.

  In the normal reciprocating scan, as shown in FIG. 6, a scan of one line L is performed in the forward path, and a scan of the next line L is performed by changing the line in the return path. Then, the luminance is sequentially detected for each region P by the photodetector 45, and the image data of the sample S is acquired.

  On the other hand, in this embodiment, as shown in FIG. 5, one line L is scanned in the forward path (indicated by the thin line R1) and the backward path (indicated by the thick line R2). Although the forward path R1 and the backward path R2 are displayed differently in FIG. 5, the center position of the same line L is actually scanned. Then, the luminance is sequentially detected for each region P by the photodetector 45, and the image data of the sample S is acquired. The image data of the sample S is acquired separately as the image data of the forward path R1 and the image data of the return path R2. The reciprocating scan is performed by scanning the forward path with light having a low laser power and scanning the backward path with light having a high laser power without changing the gain of the photodetector 45. That is, at the conversion point C1 from the forward path R1 to the return path R2, the laser light source 23 is controlled by the CPU 51, and the laser power light is changed from weak light to strong light. On the contrary, at the conversion point C2 from the return path R2 to the forward path R1, the laser power light is changed from strong light to strong light.

  In this way, by scanning the forward path R1 with light having a low laser power, the fluorescence of the sample S does not remain up to the return path R2, and the detection accuracy can be improved. Further, by scanning the forward path R1 of the same line L with light having a low laser power and scanning the return path R2 with light having a high laser power, it becomes possible to acquire image data having different laser powers at short time intervals. The so-called live cell in which the state of the sample S changes with time can also be handled.

  Step S2: A first image G1 is created from the image data obtained by the forward path R1 scan, and a second image G2 is created from the image data obtained by the backward path R2 scan. Since the first image G1 is an image obtained by a forward scan with a low laser power, the overall brightness value is low as shown in FIG. FIG. 7 is an ideal image corresponding to the schematic sample S of FIG. 5, and an actual image is an image G1 ′ as shown in FIG. 8, for example. Since the second image G2 is an image obtained by a backward scan with a strong laser power, the overall brightness value is high as shown in FIG. 9 is an ideal image corresponding to the schematic sample S of FIG. 5, and an actual image is an image G2 'as shown in FIG. 10, for example.

  Step S3: The CPU 51 determines whether or not there is a saturated pixel H in the second image G2. That is, since the second image G2 is an image obtained by scanning the return path R2 where the laser power is strong, the overall brightness value is high as shown in FIG. There are relatively many pixels H having luminance values corresponding to the luminance values. Therefore, if such a pixel H is left as it is in the second image G2, an image different from the actual image of the sample S is obtained, and a high-quality image cannot be obtained.

  FIG. 11 shows the relationship between the intensity of light input to the photodetector 45 and the output value output from the photodetector 45. A straight line a indicates a case where the laser power is weak, and a straight line b-b ′ indicates a case where the laser power is strong. When the gain of the photodetector 45 is the same, the S / N of the output value A of the portion where the intensity of the input light is weak is deteriorated. Further, when the intensity of the laser power is large, the intensity of the light input to the photodetector 45 becomes large and becomes saturated at a predetermined output value Max. Accordingly, it is impossible to know the exact output value of the wavy portion b 'of the straight line b-b'.

  Step S4: When there is a saturated pixel H in the second image G2, the luminance value of the pixel of the second image G2 is corrected based on the luminance value of the corresponding pixel of the first image G1. First, when there is a saturated pixel H in the second image G2, the CPU 51 extracts a pixel corresponding to the saturated pixel H from the pixels of the first image G1. Then, the luminance value of the pixel of the second image G2 is corrected based on the extracted luminance value of the pixel.

  For example, when the gain of the photodetector 45 is the same, the intensity of light input to the photodetector 45 from the same region P of the sample S increases in proportion to the intensity of the laser power to be irradiated. Accordingly, as shown in FIG. 5, assuming that the intensity of the light having a low laser power used for the scan in the forward path R1 is I1, and the intensity of the light having a high laser power used for the scan in the return path R2 is I2, the scan in the backward path Then, compared with the forward scan, light having an intensity of I = I2 / I1 times obtained by dividing I2 by I1 is input to the photodetector 45. Therefore, in this embodiment, the luminance value of the pixel extracted from the first image G1 is multiplied by the value I (power ratio) obtained by dividing the intensity I2 of the backward scanning light by the intensity I1 of the outgoing scanning light. The luminance value of the pixel H in the saturated state of the second image G2 is corrected. Thereby, the luminance value of the saturated pixel H can be reliably corrected to a value close to the actual luminance value.

  That is, as indicated by the straight line b-b 'in FIG. 11, when the intensity of the laser power is large, the intensity of the light input to the photodetector 45 is increased and saturated at a predetermined output value. However, as shown in FIG. 12, for example, by multiplying the output value D of the point d on the straight line a by I to make the point d ′ on the extension of the straight line b, the wavy line b ′ saturated in FIG. The luminance value of the part can be obtained reliably.

  FIG. 13 shows a corrected image G3 obtained by correcting the second image G2 with the first image G1. In the corrected image G3, as shown in FIG. 14, the luminance value of the pixel H in the saturated state (the region surrounded by the square of V1 in the figure) is corrected. In FIG. 14, the luminance value of each pixel of the image data scanned in the return path is displayed as V2 (i, j). When the brightness value V2 (i, j) of the pixel of the image data scanned in the backward path is a saturated value, the brightness value of the pixel is I times the brightness value of the image data acquired in the forward path scan. The corrected luminance value is corrected to V1 (i, j). 13 is an ideal image corresponding to the schematic sample S in FIG. 5, and an actual image is an image G3 ′ as shown in FIG. 15, for example. This image G3 'is an image in which the photodetection dynamic range is expanded as compared with the images G1' and G2 'shown in FIGS.

  Step S5: If the image is corrected in step S4, the corrected image G3 is output to the monitor 21. The observer can observe the sample S by looking at the image G3 displayed on the monitor 21 or printing the image G3. If there is no saturated pixel in the second image G2 in step S3, the second image G2 is output to the monitor 21.

In the scanning confocal microscope described above, it is determined whether or not there is a saturated pixel H in the second image G2 obtained by the backward scan with a strong laser power, and the saturated pixel H is determined as the laser power. Since the correction is made using the luminance value of the corresponding pixel of the first image G1 obtained by the scan of the weak outbound path, the sample S having a large luminance difference in the displayed image is displayed as one image. It can be observed reliably.
(Supplementary items of the embodiment)
As mentioned above, although this invention was demonstrated by embodiment mentioned above, the technical scope of this invention is not limited to embodiment mentioned above, For example, the following forms may be sufficient.

  (1) In the above-described embodiment, the specific value of I, which is a value obtained by dividing the intensity I2 of the backward scanning light by the intensity I1 of the outgoing scanning light, is not particularly limited. By making it a power, it becomes possible to correct the image data only by the bit shift operation when the luminance value of the image data acquired by the forward scan is multiplied by I, and the correction can be performed at high speed.

  (2) In the above-described embodiment, an example in which correction is performed using the pixel of the first image G1 when the pixel of the second image G2 is saturated has been described. However, the pixel of the first image G1 is described. Whether or not to use the luminance value may be selected by a threshold value. For example, when there is a lot of shot noise, an upper limit and a lower limit are set, and within the range, the luminance value of the pixel of the first image G1 may be used.

  (3) In the above-described embodiment, the example in which the luminance value of the pixel of the second image G2 to be corrected is set to the saturation luminance of the photodetector 45 has been described. However, the value is set to a value lower than the saturation luminance. Also good.

DESCRIPTION OF SYMBOLS 11 ... Illumination optical system, 13 ... Scanning optical system, 15 ... Imaging optical system, 16 ... Stage, 17 ... Detection optical system, 19 ... Control part, 21 ... Monitor, 23 ... Laser light source, 27 ... Two-dimensional scanning mechanism, S: Sample.

Claims (5)

A light source capable of outputting first light and second light having a higher intensity than the first light;
Scanning means for irradiating the sample with the first light from the light source in the forward path or the second light on the return path, and scanning the sample;
Light detecting means for detecting light from the sample by the scanning;
An image processing unit that acquires an image composed of a plurality of pixels based on the light detected by the light detection unit and performs image processing on the acquired image;
The image processing means includes
Determining means for determining whether or not a pixel of the second image acquired by irradiating the sample with the second light is a pixel having a predetermined luminance or higher;
Correction means for correcting a luminance value of a pixel equal to or higher than a predetermined luminance of the second image based on a luminance value of a corresponding pixel of the first image acquired by irradiating the sample of the first light;
A scanning confocal microscope characterized by comprising: The scanning confocal microscope according to claim 1,
The scanning confocal microscope, wherein the predetermined luminance is a saturation luminance of the light detection means. The scanning confocal microscope according to claim 1 or 2,
The correction means multiplies the luminance value of the corresponding pixel of the first image by a value obtained by dividing the intensity of the second light by the intensity of the first light, and A scanning confocal microscope characterized by correcting a luminance value of a pixel. The scanning confocal microscope according to any one of claims 1 to 3,
The scanning confocal microscope characterized in that the scanning means reciprocally scans the same portion of the sample. The scanning confocal microscope according to claim 4,
The scanning confocal microscope characterized in that the scanning means scans the sample with the first light in the forward path and scans the sample with the second light in the backward path.