JP2009300520A - Living body observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a living body observation device capable of providing a fine image at an arbitrary timing. <P>SOLUTION: The living body observation device comprises: a light source part for emitting an excitation light beam; a scanning optical system for scanning the excitation light beam; an objective lens for irradiating a living body sample with the excitation light beam; and a living body contact surface that contacts the living body sample. The living body observation device further comprises: marker assignment means in which a marker which can be restored based on the difference between the distance of a marker fragment in a first scanning line by the scanning optical system and the distance of a marker fragment in a second scanning line is disposed on the living body contact surface; a detection optical system for detecting detection light from the living body sample; an image display means for displaying an electric signal outputted from the detection optical system as image data; and an image reconfiguration means for reconfiguring the image data displayed by the image display means based on the position information of the marker. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a living body observation apparatus having an image reconstruction function for obtaining a still image from a vibrating living body.

  In order to analyze biological functions and elucidate pathology, there is an increasing interest in techniques for observing living bodies. However, because the living body is alive, it has vibration due to breathing, pulsation, and pulsation. This vibration is a very big problem as a factor that obstructs observation, such as blurring of the observation image.

As a device for observing such a dynamic behavior, a microscope image pickup device is known (for example, Patent Document 1).
JP-A-2005-274783

  However, such a conventional microscopic image pickup device detects a movement of a living body and extracts only an image when the living body is not moving, thereby obtaining an image free from image blur due to a living body vibration. Images can only be captured at the moment when the living body is not moving.

  Furthermore, in anesthetized mice, heart rate occurs about 10 times per second and breathing occurs about once per second. In the mouse liver, vibration due to heartbeat has an amplitude of about 300 μm, and vibration due to respiration has an amplitude of about 2 mm. When attempting to observe such a living body with the magnifying optical system, image blur always occurs due to vibration of the living body. That is, the conventional technology has a problem that not only a living body for an arbitrary time can be observed, but also a fine image cannot be obtained when performing detailed observation by enlarging.

  Accordingly, an object of the present invention is to provide a living body observation apparatus that can obtain a fine image at an arbitrary timing.

  As a result of earnest research, the present inventor has newly provided a marker providing means in the living body observation apparatus and reconstructed an image based on the marker information, thereby solving the above problem.

  That is, the present invention relates to a light source unit for emitting an excitation light beam, a scanning optical system for scanning the excitation light beam, an objective lens for irradiating the biological sample with the excitation light beam, and the living body A marker having a living body contact surface in contact with a sample and capable of being restored based on a difference between a marker fragment distance in the first scanning line and a marker fragment distance in the second scanning line by the scanning optical system. Marker providing means, a detection optical system for detecting detection light from the biological sample, and an image display means for displaying an electrical signal output from the detection optical system as image data, disposed on the biological contact surface. There is provided a living body observation apparatus comprising: an image reconstruction unit that reconstructs image data displayed by the image display unit based on position information of the marker.

  The present invention can provide a living body observation apparatus that can obtain a fine image at an arbitrary timing.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the embodiments described below are merely examples for explaining the configuration of the present invention in detail. Accordingly, the present invention should not be construed as being limited based on the description given in the following embodiments. The scope of the present invention includes all embodiments including various modifications and improvements of the following embodiments as long as they are within the scope of the invention described in the claims.

<First Embodiment>
FIG.
FIG. 1 is a schematic diagram of a living body observation apparatus 100 in the first embodiment. The biological observation apparatus 100 of the present invention includes a light source unit 110 for emitting an excitation light beam, a scanning optical system 120 for scanning the excitation light beam, and an objective for irradiating the biological sample with the excitation light beam. Based on the difference between the distance of the marker fragment in the first scanning line by the scanning optical system and the distance of the marker fragment in the second scanning line, which has a lens 130 and a biological contact surface in contact with the biological sample. The marker providing means 140 in which a marker that can be restored is arranged on the biological contact surface, the detection optical system 150 that detects detection light from the biological sample, and the electrical signal output from the detection optical system The image display means 160 displayed as data, and the image reconstruction means 160 which reconfigure | reconstructs the image data displayed by the said image display means based on the positional information on the said marker are provided.

  The light source unit 110 includes a laser light source 111 that emits excitation light, an optical fiber 112 (multimode fiber) that transmits the excitation light emitted from the laser light source 111, and the excitation light transmitted by the optical fiber 112 as an excitation light beam. And an injection part 113 for injection. The excitation light beam emitted from the emission unit 113 is collected by the condenser lens 171, reflected by the dichroic mirrors 172 and 173, and enters the scanning optical system 120.

  The scanning optical system 120 is configured using a known scanning element that scans a light beam such as a galvanometer mirror (XY galvanometer mirror unit) or an acoustooptic device. The excitation light that has entered the scanning optical system 120 is deflected, enters the objective lens 130 through the projection lens 174 and the imaging lens 175, and the biological sample A is irradiated with the excitation light beam through the objective lens 130. Detection light (fluorescence and reflected light) from the biological sample A is returned through the same path, passes through the dichroic mirror 173, is collected on the condenser lens 176, and then enters the detection optical system 150.

  The marker imparting means 140 has a living body contact surface 141 and is formed of a known member capable of placing a marker on the living body contact surface 141, for example, a stabilizer or a cover glass. In particular, when a stabilizer is used, it is possible to prevent image information from being lost due to image shift in the optical axis direction by suppressing vibration of the living body in the optical axis direction (Z-axis direction).

  When a stabilizer is used, the material of the biological contact surface and the back surface of the marker may be changed in order to reduce sliding friction with the glass disposed on the biological contact surface of the stabilizer. In this case, the surface in contact with the living body is compatible with the living body and easily adheres to the glass, and an optically transparent slidable resin may be used on the back surface. Alternatively, the glass disposed on the biological contact surface of the stabilizer may be changed to a resin that is optically transparent and has good slidability. Moreover, you may give the coating which improves slidability to the other side of a biological contact surface. Alternatively, an optically transparent resin having good slidability may be fixed to the mouse using a biological adhesive.

  The biological sample A is placed on the stage 190. In order to observe a desired position of the biological sample A, the stage 190 is preferably composed of a rotary stage and an XY stage.

  The detection optical system 150 includes an incident unit 151 that allows detection light (fluorescence or reflected light) from a biological sample to enter, an optical fiber 152 (multimode fiber) that transmits the detection light incident on the incident unit 151, and an optical fiber 152. A detector 153 that converts the detection light transmitted by the light into an electric signal reflecting the light intensity and outputs the electric signal.

  The image display means 160 includes an arithmetic processing unit 161 that converts the electrical signal output from the detector 153 into pixel information that constitutes an image, and a monitor 162 that displays the result of the arithmetic processing as an image. The arithmetic processing unit 161 acquires electrical signals sequentially obtained by scanning the biological sample A by the scanning optical system 120 as pixel information corresponding to the position where the electrical signals are obtained, and sequentially determines the luminance of each pixel. As a result, the biological image in the scanning range of the biological sample A is displayed on the monitor 162.

  At this time, since the biological sample A constantly vibrates, the image data displayed by the image display means 160 is shifted in the sub-scanning direction. Here, the sub-scanning direction is a direction perpendicular to the scanning direction. For example, when scanning is performed in the X-axis direction on the XY plane, the Y-axis direction is the sub-scanning direction. The scanning optical system 120 scans in the X-axis direction (first scan), moves in the Y-axis direction by the width of the first scan, and then scans in the X-axis direction (second scan). ). Since scanning in the X-axis direction is performed in a very short time, the living body image obtained by each scanning is hardly blurred. However, a time lag occurs between the first scan and the second scan, and the biological image is shifted due to the time lag. The image reconstruction unit 160 corrects the deviation of the biological image, more specifically, the deviation of the image generated between the first scanning line and the second scanning line due to the biological vibration based on the marker information. This is a means for restoring the correct positional relationship.

  The image reconstruction unit 160 includes an arithmetic processing unit 161 that reconstructs image data information displayed by the image display unit 160 based on marker position information, and a monitor that displays an image reconstructed as a result of the arithmetic processing. 162. Note that the image display unit 160 and the image reconstruction unit 160 may be constructed in the same PC, and both can be configured by the arithmetic processing unit 161 and the monitor 162.

FIG.
FIG. 2 is a schematic diagram illustrating the biological contact surface 141 of the marker applying unit 140.

The marker 1 is disposed on the biological contact surface 141 of the marker applying unit 140. Marker 1 is a distance d ₁ of the marker fragments in the first scan line, they are arranged in a recoverable form on the basis of the difference between the distance d ₂ of the marker fragments in the second scan line.

  Here, the first scanning line means an arbitrary scanning line by the scanning optical system 120 (illustrated as the scanning line 10 in the figure), and the second scanning line is the same as the scanning optical system 120. Means a scan line that is different from the first scan line and is illustratively represented as scan line 20 in the figure. None of them means a specific scanning line, and represents the relationship between the nth scanning line (n is a natural number) and an arbitrary scanning line other than the nth scanning line. In the following drawings, for example, the X-axis direction is represented as a scanning direction, and the sub-scanning direction is represented as a Y-axis direction.

The marker fragment distance d ₁ means the distance between the edges of the marker in the first scanning line 10, and the marker fragment distance d ₂ means the distance between the marker edges in the second scanning line 20. Here, if the marker 1 is arranged so that the distance d ₁ and the distance d ₂ are different, the position information of the first scanning line 10 and the second scanning line 20 is the distance between the marker fragment having the distance d ₁ and the distance d. Stored by marker fragment with ₂ . That is, when a deviation occurs between the first scanning line 10 and the second scanning line 20 due to the vibration of the living body, the marker fragment having the distance d ₁ on the first scanning line 10 and the second scanning Based on the marker fragment having the distance d ₂ on the line 20, the relative positional relationship between the first scanning line 10 and the second scanning line 20 can be restored. As a result, it is possible to restore an image in which image blur has occurred due to vibration of the living body and reproduce it as a fine still image.

Further, as described later, including the auxiliary marker, if multiple markers are given, sometimes to define the distance between each of the markers as d _n. What is important is that the marker information on each scanning line can be distinguished from the marker information on other scanning lines. If the marker information is only to be saved to the positional information of each scanning line, and there is no particular limitation on the definition of the method of the distance d _n.

The shape of the marker is not particularly limited as long as the above-described requirements are satisfied. For example, since the right triangular marker shown in FIG. 2 has an inclination BB with respect to the scanning direction, the marker fragment distance d ₁ in the first scanning line and the marker fragment in the second scanning line. The distance d ₂ is always different. Therefore, since the position information of all the scanning lines is stored according to the distance of the marker fragment, the position of each scanning line is adjusted so that the shape of the marker is restored when a deviation occurs between the scanning lines due to biological vibration. By doing so, a fine still image can be restored. As other marker shapes, for example, polygonal markers such as quadrangular, pentagonal, hexagonal, heptagonal, octagonal, etc., and linear or curved markers can be used.

  There is no restriction | limiting in particular in the kind of marker 1, All the markers well-known at the time of application can be used.

  For example, first, non-fluorescent and non-reflective markers can be used. The non-fluorescent and non-reflective marker can prevent the reflection of intense light emitted from the marker on the fluorescence observation image of the living body, and does not use a laser for imaging the marker. A plurality of fluorescence can be freely selected. In this case, since the marker part is black in the observation image, the position information of the edge of the marker can be easily extracted.

  Second, the reflective film can be used as a marker. Since the reflection film marker can be detected at the same wavelength as the excitation light for living body observation, a laser for exciting the marker is unnecessary, and a plurality of observation fluorescence can be freely selected accordingly. In addition, since the reflection efficiency is one order higher than the fluorescence excitation efficiency, the marker can be imaged clearly. As a result, it becomes easier to detect the edge, and the image can be reconstructed with high accuracy.

  Third, a fluorescent material can be used as a marker. If a fluorescent material having a wavelength different from that for fluorescence for living body observation is used as a marker, observation is not hindered. Furthermore, the fluorescent material used as the marker may have the same wavelength as the fluorescence for living body observation. In this case, the fluorescence intensity of the marker is set stronger than the observation fluorescence intensity. Then, binarization is performed using a level brighter than the fluorescence of the living body and darker than the fluorescence of the marker as a threshold, and the position information of the edge of the marker can be easily extracted from the binarized image. In this case, many types of fluorescence can be used for living body observation without reducing the types of fluorescence that can be used for observation.

FIG.
FIG. 3A is an image displayed by the image display means 160. Due to the time lag between the first scan 10 and the second scan 20, the position information of each scan line is shifted.

FIG. 3B is an image reconstructed by the image reconstruction unit 160 based on the position information of the marker 1. The relative positional relationship between the first scanning line 10 and the second scanning line 20 is specified based on the positional information of the marker fragment distances d ₁ and d ₂ . Then, by moving and adjusting the positional relationship between the first scanning line 10 and the second scanning line 20 so that the shape of the marker is restored based on the specified position information, fine stillness without image blurring is achieved. Images can be restored. It should be noted that the first and second scanning lines here do not mean specific scanning lines, but represent arbitrary two scanning lines.

  Here, the image reconstructing means 160 adjusts the movement of each scanning line, from the left end (CC line in FIG. 3B) having the maximum value among the left ends of all scanning lines, to the minimum value among the right ends of all scanning lines. Up to the right end (D-D line in FIG. 3B) having a is displayed as an image. As a result, the image reconstructed by the image reconstructing means 160 does not lack image information, and a complete biological image can be provided without providing supplementary image information.

  The monitor 162 may display the best position of the marker 1 lightly. The observer can restore a clear still image from the blurred biological image by correcting the positional deviation for each scan line based on the position information of the marker 1 displayed lightly.

FIG.
FIG. 4 is a flowchart of the arithmetic processing performed by the image reconstruction unit 160.

  When the fluorescent material used as a marker has the same wavelength as the fluorescence for living body observation in the living body observation using fluorescence, first, it is binarized using a level brighter than the fluorescence of the living body and darker than the fluorescence of the marker as a threshold. A valued image is obtained (31). The edge position of the marker 1 can be detected from this binarized image.

  Next, the position information of the edge of the marker 1 is extracted (32). Then, an edge-to-edge distance is calculated from the extracted position information (33).

  The calculated distance between the edges is collated with the shape information of the marker 1 stored in advance, and the position in the y-axis direction is determined (34). Then, the position information in the x-axis direction is determined by collating with the position information of the marker 1 stored in advance (35).

  An image can be reconstructed by performing the above steps for each scan.

  When a non-fluorescent and non-reflective marker is used, or when a fluorescent material having a wavelength different from fluorescence for living body observation is used as a marker, the binarization step (31) is unnecessary, and the subsequent steps are as follows. All are the same.

FIG.
FIG. 5 is a flowchart of a calculation process performed by the image reconstruction unit 160 for correcting the orientation of the marker.

  When the marker shape is a polygon, it is necessary to correct the orientation of the marker using pattern matching from the marker shape stored in advance.

  After acquiring the binarized image (41), the stored marker (mask pattern) is read (42). Next, the mask pattern is rotated (43) and superimposed with the binarized image (44). At this time, it is determined whether or not the orientation of the mask of the binarized image matches the orientation of the mask of the mask pattern (45). If not, the mask pattern is rotated again (43), 2 The digitized images are overlaid (44).

  By performing the above arithmetic processing, a still image can be restored in the correct orientation.

FIG.
FIG. 6 is a schematic diagram showing the biological contact surface 141 on which auxiliary markers (calibration marks) are arranged.

  In FIG. 6, one linear marker 1 that diagonally crosses the biological contact surface 141 is provided, and a plurality of auxiliary markers 2 are provided over the entire biological contact surface 141.

Since the marker 1 of the present invention bears the position information of each scanning line, it needs to be large enough to be reflected in the entire observation field. In this regard, a marker constituted by a straight line or a curve is excellent because it can be easily configured so as to be reflected in the entire observation field even though the marker area is small. However, for example, in a marker constituted by a straight line, the distance d ₁ of the marker fragment in the first scanning line 10 is the same as the distance d ₂ of the marker fragment in the second scanning line 20, and the position of each scanning line Cannot carry information. Accordingly, it is necessary to provide one or more auxiliary markers 2 (calibration marks) so that the linear marker can carry position information of each scanning line. When the auxiliary marker 2 makes the marker fragment distance in the first scanning line 10 different from the marker fragment distance in the second scanning line 20, the image can be reconstructed by the linear marker 1.

Here, the distance between the marker fragments in each scanning line does not mean the width of the linear marker 1 in the X-axis direction, but the distance information between the markers existing in each scanning line including the auxiliary marker 2. means. For example, in FIG. 6, the auxiliary marker 2 is arranged uniformly over the entire surface of the biological contact surface 141. At this time, the distance of the marker fragment in the first scanning line 10 means distance information defined by d _1-1 , d _1-2 , and d _1-3 . In addition, the distance between the marker fragments on the second scanning line 20 means distance information defined by d _2-1 , d _2-2 , and d _2-3 . These distance information can be identified, and as a result, these distance information can carry position information on the living body contact surface 141 of the first scanning line 10 and the second scanning line 20. Basically, it is preferable that the auxiliary marker 2 is arranged so as to enter two or more in each scanning line.

FIG.
FIG. 7 is a schematic diagram showing the biological contact surface 141 in which linear markers having different thicknesses are arranged in addition to the auxiliary marker 2.

  In FIG. 7, in addition to the auxiliary marker 2, linear markers 1 b and 1 c having different thicknesses are arranged in a direction intersecting with the linear marker 1. In this way, in addition to the auxiliary marker 2, each scanning line is configured to include two or more lines, so that the position information given by the marker fragment of each scanning line becomes clearer and more reliably and easily. Still images can be reconstructed. Basically, the marker 1 is a thin line arranged in a lattice shape (diamonds arranged in a line) that are not orthogonal to each other, each scanning line is arranged so that two or more lines are included, and in the scanning direction. It is preferable that they are arranged with an inclination. Also in this case, as described above, the distance between the marker fragments means position information given by the distances between a plurality of markers scattered on each scanning line.

  The lines of the marker 1 can be distinguished from each other in a range corresponding to the displacement amount of the vibration of the living body (for example, different thickness, different color, different excitation efficiency, different reflection efficiency, etc.). ). For example, if there is a vibration of 10 μm, the same pattern may be repeated at 10 μm intervals.

  The marker interval is defined so that at least one marker is included in the correction unit. For example, when an image of 512 × 512 is obtained over 0.1 sec for one line scan, the pixels that can be scanned within a time when the image is not blurred (allowable) due to vibration of the living body are corrected as one unit. For example, the number of pixels corresponding to 1 msec (here, 5 pixels) is corrected together. In this case, the marker interval is defined so that one or more marker lines are included in 1 × 5 pixels. It should be noted that an image may be complemented with an average value of adjacent pixels in a portion where an image is not captured due to inhibition by the marker.

FIG.
FIG. 8 is a schematic diagram showing a state in which a missing part of image information is complemented by different imaging data.

  When the vibration of the living body in the optical axis direction (Z-axis direction) is extremely small, or even when the vibration of the living body in the Z-axis direction is recognized, the vibration of the living body in the Z-axis direction is sufficient by the marker applying means 140 such as a stabilizer. In theory, all of the imaging data is in focus, and a complete still image can be reconstructed only by the correction in the XY directions described above.

  However, when the vibration of the living body in the Z-axis direction exists at a certain level or more, depending on the position of the living body, there is a part where the marker providing means 140 such as a stabilizer cannot be arranged. Scan lines that are not in focus are generated due to vibration in the direction. When this unfocused scanning line is used as image information, image blurring occurs and a fine still image cannot be obtained. Therefore, in order to obtain a fine still image, it is necessary to complement the missing image information by the second imaging without focusing on the first imaging.

  FIG. 8A shows the first image data. The image reconstruction unit 160 reconstructs an image of the living body image (A-1) in which the image shift has occurred for each scanning line based on the position information of the marker 1 (A-2). When a region where some data does not exist in the X direction occurs, it is sufficient to extract only a region where all the data is gathered as shown in FIG. 3B. Alternatively, a method in which imaging is repeatedly performed until all the data is obtained can be considered.

At this time, the image reconstruction means 160 simultaneously identifies image information with image blur and removes it from the image information (P ₁ , P ₂ and P _{3 in} A-2). The degree of focus is determined from the sharpness of the marker edge. Then, only the image data determined to be in focus is determined as image information (A-3).

  FIG. 8B shows the second image data. Similar to the first image data, the image reconstruction unit 160 reconstructs the biological image (B-1) in which the image shift occurs for each scanning line (B-2).

In the second and subsequent image data, only the deficient data area of the first image data is set as the attention area (P ₁ , P ₂ and P ₃ areas), and the degree of focus only on the attention area. Judgment is made. By doing in this way, calculation time can be shortened. It is important to prioritize the data at the start of shooting and to use as much data as possible on the first sheet. This is because, when a user desires an image at a point of interest for a certain reaction, priority is given to information in the vicinity of the desired time as much as possible.

The image reconstruction means 160 determines the degree of focus only for the P ₁ , P _2, and P ₃ regions, identifies the P _1-2 and P _2-2 regions that are not in focus, and extracts these from the image information. Remove (B-2). Then, only the image data determined to be in focus is added to the image information (A-3) determined for the first image, and this is determined as image information (B-3).

FIG. 8C shows the third image data. Similar to the first and second image data, the image reconstructing means 160 reconstructs the biological image (C-1) in which the image shift occurs in each scanning line (Y-axis direction) (C-2). . Then, the image reconstruction means 160 determines the degree of focus only for the data areas (P _1-2 and P _2-2 areas) that are insufficient up to the second sheet. Then, the image data determined to be in focus is added to the image information (B-3) determined for the first and second images, and this is determined as image information (C-3).

  In FIG. 8, all the missing data is supplemented up to the third image data by way of example, but if all the missing data are not supplemented even in the third image, the fourth and fifth image data are continued. Completion by

  The finally obtained image of FIG. 8C-3 is a complete and fine still image in which the deficient data is supplemented with the second and subsequent imaging data while being based on the first imaging data. Even when a living body that vibrates strongly in the optical axis direction (Z-axis direction) is captured by such an image reconstruction means complementing operation of the image data, a reliable and fine still image can be obtained.

  In addition, instead of giving priority to the first data, it is possible to extract only the portion where the marker is clearly imaged from each imaged data and finally combine them at once. In this case, an average state during all imaging can be displayed. Specifically, an in-focus area is extracted from all images, and an average value is obtained for the overlapping portions of a plurality of images. By doing so, noise can also be reduced, and an effect of improving image quality can be obtained.

Schematic diagram of the biological observation apparatus 100 in the first embodiment Schematic diagram showing the biological contact surface 141 of the marker applying means 140 An image displayed by the image display means 160 (FIG. 3A) and an image reconstructed by the image reconstruction means 160 based on the position information of the marker 1 (FIG. 3B) Flowchart of arithmetic processing performed by image reconstruction means 160 Flowchart of arithmetic processing performed by the image reconstruction means 160 for correcting the direction of the marker Schematic diagram showing the biological contact surface 141 on which an auxiliary marker (calibration mark) is arranged The schematic diagram showing the biological body contact surface 141 in which the linear marker from which thickness differs in addition to the auxiliary marker 2 is arrange | positioned Schematic diagram showing how the missing part of image information is complemented by different imaging data

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Marker, 1b, 1c ... Linear marker of different thickness, 2 ... Auxiliary marker, 10 ... 1st scanning line, 20 ... 2nd scanning line, 100 ... Biological observation apparatus of the present invention, 110 ... light source unit, 111 ... laser light source, 112, 152 ... optical fiber, 113 ... ejecting unit, 120 ... scanning optical system, 130 ... objective Lens: 140 ... Marker applying means, 141 ... Living body contact surface, 150 ... Detection optical system, 151 ... Incident part, 153 ... Detector, 160 ... Image display means, Image replay Configuration means 161 ... arithmetic processing unit 162 ... monitor 171 176 ... condensing lens, 172, 173 ... dichroic mirror, 174 ... projection lens, 175 ... imaging lens .

Claims (8)

A light source unit for emitting an excitation light beam;
A scanning optical system for scanning the excitation light beam;
An objective lens for irradiating the biological sample with the excitation light beam;
It has a biological contact surface in contact with the biological sample, and can be restored based on the difference between the marker fragment distance on the first scanning line and the marker fragment distance on the second scanning line by the scanning optical system. A marker providing means in which a marker is disposed on the biological contact surface;
A detection optical system for detecting detection light from the biological sample;
Image display means for displaying the electrical signal output from the detection optical system as image data;
A living body observation apparatus comprising: image reconstruction means for reconstructing image data displayed by the image display means based on position information of the marker.   The marker is formed of a polygon, and the polygon is arranged such that the distance between the marker fragments in the first scanning line is different from the distance between the marker fragments in the second scanning line. The biological observation apparatus described in 1.   The marker is formed of a straight line or a curve, and an auxiliary marker for making the distance of the marker fragment in the first scanning line different from the distance of the marker fragment in the second scanning line is around the straight line or the curve. The living body observation apparatus according to claim 1, which is arranged. The marker applying means is composed of a stabilizer or a cover glass,
The living body observation apparatus according to any one of claims 1 to 3, wherein one or more of the markers are provided on the living body contact surface of the stabilizer or the cover glass. A light source unit for emitting an excitation light beam;
A scanning optical system for scanning the excitation light beam;
An objective lens for irradiating the biological sample with the excitation light beam;
It has a biological contact surface in contact with the biological sample, and can be restored based on the difference between the marker fragment distance on the first scanning line and the marker fragment distance on the second scanning line by the scanning optical system. A marker providing means in which a marker is disposed on the biological contact surface;
A detection optical system for detecting detection light from the biological sample;
Image display means for displaying the electrical signal output from the detection optical system as image data;
An image processing method using a biological observation apparatus, comprising image reconstruction means for reconstructing image data displayed by the image display means based on position information of the marker,
Determining the distance of each marker fragment in each scan line;
And reconstructing an image by moving and adjusting each scanning line based on the determined distance information.   The image processing method according to claim 5, wherein the step of reconstructing the image includes a first step of restoring a marker and a second step of correcting the orientation of the restored marker. The first step comprises:
Extracting the position information of the edge of the marker;
Calculating a distance between the edges;
Determining a position in the y direction from the stored marker shape information;
The image processing method according to claim 6, further comprising: determining a position in the x direction from the stored marker position information. The second step comprises:
Reading the stored marker;
Rotating the marker reconstructed by the first step;
Superimposing the reconstructed marker with the stored marker;
The image processing method according to claim 6, further comprising a step of determining whether or not the direction of the reconstructed marker matches the direction of the stored marker.

Images