JP5074098B2 - Scanning optical device - Google Patents

Description

  The present invention relates to a scanning optical device, and more particularly to a multi-photon excitation scanning optical device using a multi-photon excitation phenomenon.

Conventionally, a scanning optical device that acquires a bright fluorescence image having a high resolution in the depth direction of the sample by using the multiphoton excitation phenomenon is known, but since the fluorescence generated from the sample is scattered by the sample, There is a problem that a fluorescent image based on fluorescence generated at a deep position of the sample is darker than a fluorescent image based on fluorescence generated at a shallow position of the sample.
In order to solve this problem, for example, the invention disclosed in Patent Document 1 corrects fluctuations in apparent brightness on the screen by performing image processing on the acquired image data.

JP 2000-275541 A

  However, when the acquired image data is corrected by performing image processing, there is a disadvantage that the amount of luminance information contained in the image data itself is small and the fluorescent image obtained by the correction becomes unclear. For example, it is acquired by adjusting the light source, optical scanning unit or photodetector so that the fluorescence image based on the fluorescence generated from the shallow position of the sample becomes clear and detecting the fluorescence generated from the deep position of the sample Since the fluorescent image thus formed is dark overall and has little luminance information, a clear image cannot be constructed even if the brightness is corrected by image processing.

Conversely, by adjusting the light source, optical scanning unit or photodetector so that the fluorescence image based on the fluorescence generated from the deep position of the sample becomes clear, the fluorescence generated from the shallow position of the sample is detected. The acquired fluorescent image is saturated in luminance, and similarly, even if the brightness is corrected by image processing, a clear image cannot be constructed.
That is, in order to acquire a clear fluorescent image regardless of the depth of the sample, it is necessary to acquire a fluorescent image having the same brightness without performing image processing.

  The present invention has been made in view of the above-described circumstances, and in order to acquire a clear fluorescent image regardless of the depth of the sample, a fluorescent image having the same brightness is acquired without performing image processing. It is an object of the present invention to provide a scanning optical device that can be used.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a multi-photon excitation light source that emits an ultra-short pulse laser beam, a scanning unit that scans an ultra-short pulse laser beam emitted from the multi-photon excitation light source on a sample, and a scanning unit that scans the sample. A focusing depth adjusting unit that adjusts the depth of the focusing position of the ultrashort pulse laser beam in the sample, and a fluorescence detecting unit that detects the fluorescence emitted from the focusing position of the ultrashort pulse laser beam in the sample And an index from a multiphoton fluorescence image acquired from a predetermined depth position based on the luminance information of the fluorescence detected by the fluorescence detection unit and the scanning position of the ultrashort pulse laser beam by the scanning unit A multi-photon fluorescence image acquired on the basis of a reference luminance condition setting unit for setting a reference luminance condition , luminance information of the fluorescence detected by the fluorescence detection unit, and a scanning position of the ultrashort pulse laser beam by the scanning unit of Degree condition, so that almost the same as ultrashort pulsed laser light condensing said reference luminance condition set by the reference luminance condition setting unit for each depth position within the sample, the focusing depth adjusting section The multi-photon excitation light source, the hardware setting adjustment unit that adjusts at least one hardware setting value of the scanning unit or the fluorescence detection unit, and the luminance condition of the multi-photon fluorescence image are substantially the same. Hardware that stores the at least one hardware setting value of the light source for multiphoton excitation, the scanning unit, or the fluorescence detecting unit in association with a plurality of different depths of the condensing position of the ultrashort pulse laser beam in the sample comprises a ware set value storage unit, wherein using the stored hardware settings, and a control unit for acquiring the multiphoton fluorescence image of the sample over a desired depth range, a scanning optical To provide a location.

  According to the present invention, the ultrashort pulse laser beam emitted from the multiphoton excitation light source is condensed at the depth position of the sample adjusted by the concentration depth adjusting unit, so that Multiphoton fluorescence is generated by the multiphoton excitation phenomenon. Then, the scanning unit scans the condensing position of the ultrashort pulse laser beam on the sample, thereby generating multiphoton fluorescence at each scanning position and detecting it by the fluorescence detection unit. Therefore, a multiphoton fluorescence image can be acquired based on the luminance information of the fluorescence detected by the fluorescence detection unit and the scanning position of the ultrashort pulse laser beam by the scanning unit.

  In this case, according to the present invention, the operation of the hardware setting adjustment unit causes the luminance conditions of the multiphoton fluorescence image to be substantially the same for each depth of the condensing position in the sample of the ultrashort pulse laser light. , At least one set value of the light source for multiphoton excitation, the scanning unit, or the fluorescence detection unit is adjusted. As a result, it is possible to acquire a multiphoton fluorescence image having almost the same luminance condition regardless of the depth of the condensing position of the ultrashort pulse laser light without performing image processing.

In the above invention, before Symbol hardware settings adjuster portion, a plurality of setting values stored in the hardware set value storage unit by interpolation calculation, it is also possible to adjust the set value.

  By doing in this way, the setting value storage unit is obtained by performing an interpolation operation using the setting values stored in the setting value storage unit in association with a plurality of depths with different condensing positions of the ultrashort pulse laser beam. The multi-photon excitation light source capable of acquiring a multi-photon fluorescence image having substantially the same luminance condition at a depth position other than the depth of the condensing position stored in One set value can be obtained.

Moreover, in the said invention, it is good also as providing the luminance condition setting part which sets the luminance conditions of the said multiphoton fluorescence image based on the acquired image.
Since the brightness condition of the multiphoton fluorescence image to be acquired is different for each observer, the brightness condition of the multiphoton fluorescence image is set based on the desired image by the operation of the brightness condition setting unit. It becomes possible to acquire a multiphoton fluorescence image under a luminance condition.

In the above invention, the luminance condition of the multiphoton fluorescence image may be an average luminance value of pixels having a luminance equal to or higher than a predetermined threshold value.
In this way, the influence of pixels with low luminance values can be removed, and the bright multiphoton fluorescence image becomes dark overall, or the high brightness portion of the multiphoton fluorescence image with many dark portions becomes excessively bright. Therefore, a clear image can be acquired.

In the above invention, the luminance condition of the multi-photon fluorescence image includes a number of pixels having a luminance equal to or higher than a first threshold value and a second threshold value larger than the first threshold value. It is good also as a ratio with the pixel number of the pixel which has the above brightness | luminance.
By doing this, even if the depth of the condensing position of the ultrashort pulse laser light is different, it is possible to obtain a multiphoton fluorescence image in which the distribution ratio of pixels having a luminance equal to or higher than a predetermined brightness is equivalent. it can. As a result, it is possible to prevent the occurrence of uneven brightness in the multiphoton fluorescence image due to the different depths.

In the above invention, the second threshold value may be the maximum luminance that can be expressed in the multiphoton fluorescence image.
By doing in this way, generation | occurrence | production of the pixel with which a luminance value is saturated can be controlled, and a bright multiphoton fluorescence image can be acquired.

  According to the present invention, in order to acquire a clear fluorescent image regardless of the depth of the sample, there is an effect that a fluorescent image having an equivalent brightness can be acquired without performing image processing.

A scanning optical device 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the scanning optical device 1 according to the present embodiment includes a multiphoton excitation light source 2 that emits an ultrashort pulse laser beam, and an ultrashort pulse emitted from the multiphoton excitation light source 2. A laser intensity adjusting unit 3 that adjusts the intensity of the laser beam, a scanner (scanning unit) 4 that scans the ultrashort pulse laser beam two-dimensionally, and a pupil projection lens 5 that condenses the scanned ultrashort pulse laser beam. And the imaging lens 6, the objective lens 7 for condensing the fluorescent light generated in the specimen A while condensing the ultrashort pulse laser beam made substantially parallel by the imaging lens 6 on the specimen A, and the objective Included in the fluorescence is a dichroic mirror 8 that branches the fluorescence collected by the lens 7 and returned through the imaging lens 6 from the ultrashort pulse laser beam, and the focusing lenses 9 and 10 that collect the branched fluorescence. Blocks ultrashort pulse laser light A barrier filter 11 that, and a photodetector (fluorescence detector) 12 for detecting the fluorescence collected by the condenser lens 9 and 10.

  Further, the scanning optical apparatus 1 according to this embodiment includes a control unit 13 that controls the laser intensity adjusting unit 3, the scanner 4, and the photodetector 12, and an observer inputs various inputs to the control unit 13. And an input unit 14 for performing the above and a monitor 15 for displaying a multiphoton fluorescence image constructed by the control unit 13. The objective lens 7 is provided with a condensing position adjusting mechanism 16 that moves the objective lens 7 along the optical axis direction, and is controlled by the control unit 13.

  The ultrashort pulse laser light source 2 emits an ultrashort pulse laser beam having a predetermined intensity. The laser intensity adjusting unit 3 is composed of, for example, an acousto-optic element, and adjusts the intensity of the ultrashort pulse laser beam incident on the scanner 4 according to a command from the control unit 13.

  As shown in FIG. 2, the control unit 13 determines at least one set value (hereinafter referred to as a hardware set value) of the laser intensity adjusting unit 3, the scanner 4, or the photodetector 12. (Step S1), and the hardware setting value is set so that the set luminance condition is substantially the same for each depth of the condensing position in the specimen A of the ultrashort pulse laser beam. Calculation and storage (step S2), and using the stored hardware setting value, a multiphoton fluorescence image of the sample A over a desired depth range is acquired (step S3).

More specifically, the brightness condition is set by the control unit 13 (step S1). First, as shown in FIG. 3, the observer designates a depth range that the sample A wants to observe (step S11). A multiphoton fluorescence image at a predetermined depth position (for example, the shallowest depth position in the specified depth range) is acquired (pre-scanned) and displayed on the monitor 15 (step S12), and the observer displays the monitor 15 By adjusting the input from the input unit 14 while watching, it is adjusted to a multi-photon fluorescence image having a desired brightness, and this is used as a reference image (step S13). The control unit 13 stores the hardware setting value in this reference image (step S14). Further, the control unit 13 calculates an average luminance value of pixels having a luminance equal to or higher than a predetermined threshold among the pixels of the acquired reference image, and sets this as the luminance condition I ₀ (step S15). It has become.

Next, calculation and storage (step S2) of the hardware set values by the control unit 13, here, set values of the laser intensity adjusting unit 3 and the photodetector 12 will be described.
As shown in FIG. 4, the control unit 13 first operates the condensing position adjusting mechanism 16 so that the ultrashort pulse laser is positioned at an arbitrary position deeper than the depth position of the reference image within the specified depth range. The light condensing position is moved (step S21). Here, it is determined whether or not the intensity of the ultrashort pulse laser beam can be further increased (step S22). If the intensity can be increased, the setting value of the laser intensity adjusting unit 3 is adjusted to adjust the intensity of the ultrashort pulse laser beam. raising the (step S23), it calculates the luminance condition (step S24), and the calculated luminance condition determines whether same brightness condition I ₀ in the reference image have become (step S25) as.

If it is determined that the calculated brightness condition is not identical to the brightness condition I ₀ in the reference image, so that the step S22~S25 are repeated. If they are the same, the set values of the laser intensity adjusting unit 3 and the photodetector 12 are stored (step S26), and it is determined whether or not the current depth position is the deepest part (step S27). It is like that. If it is the deepest part, after the interpolation process is performed (step S28), the process is terminated. If it is not the deepest part, steps S21 to S27 are repeated.

On the other hand, in step S22, when the intensity of the ultrashort pulse laser beam cannot be increased any more, the applied voltage HV of the photodetector 12 is increased (step S29), and the luminance condition is calculated (step S30). luminance conditions whether identical or not the luminance condition I ₀ in the reference image is determined have become (step S31) as.

If it is determined that the calculated brightness condition is not identical to the brightness condition I ₀ in the reference image, so that the step S29~S31 are repeated.
On the other hand, in step S31, when the calculated brightness condition becomes identical to the luminance conditions I ₀ in the reference image, so that the step S26 is executed.

By such processing of the control unit 13, for example, hardware setting values (setting value (ND) of the laser intensity adjusting unit 3 and setting value (HV) of the photodetector 12) as shown in FIGS. It has come to be obtained.
That is, first, as shown in FIG. 6, it is obtained by calculating hardware setting values only at a plurality of spaced depth positions (plot positions 1 to 4), and these obtained discrete values are obtained. By interpolating the typical hardware setting values by, for example, the least square method or the like (step S28), the hardware setting values at all depth positions can be obtained as shown in FIG. .

Then, after the hardware setting values corresponding to all the depth positions are obtained in this way, the multi-photon fluorescence images for all the depth positions are acquired, so that the luminance condition is obtained at all the depth positions. Multiphoton fluorescence images having substantially the same values can be acquired.
In addition, according to the scanning optical device 1 according to the present embodiment, hardware setting values are automatically acquired at all depth positions so as to match the luminance conditions of the multiphoton fluorescence image set by the observer. Therefore, there is an advantage that a multiphoton fluorescence image having high reproducibility and no brightness unevenness for each depth can be efficiently acquired in a short time.

  In this embodiment, in order to set the luminance condition, the average luminance of pixels having a luminance equal to or higher than a predetermined threshold is obtained, and the threshold is set in advance. A luminance value appropriately selected by an observer from a diagram showing the luminance distribution of an image obtained by scanning may be set as a threshold value. Alternatively, a point or range of the image obtained by the observer may be designated, and the luminance of the designated pixel or the average luminance of the designated range may be adopted as the threshold value.

In addition, the average luminance of pixels having a luminance equal to or higher than a predetermined threshold is adopted as the luminance condition. Instead of this, two threshold values are set, and the ratio of the number of pixels having the luminance equal to or higher than each threshold is expressed by the following equation: May be set as the luminance condition.
Luminance condition = number of pixels having a luminance of threshold 2 or higher / number of pixels having a luminance of threshold 1 or higher

  For example, the luminance of the multiphoton fluorescence image may be represented by 12 bits, the maximum luminance may be set to 4095, the first threshold value may be set to 3800, and the second threshold value may be set to 4000. In this case, the luminance condition may be set so that the luminance condition = 1%.

By doing this, the hardware setting value is set so that the luminance distribution in the multiphoton fluorescence image at each depth is almost equal, and the multiphoton fluorescence image without brightness unevenness even if the depth changes. Can be acquired.
In this case, the first threshold value may be set in advance, or may be designated by an observer with reference to a diagram or the like. Alternatively, the observer may select a range or one point of the obtained image, and the minimum luminance may be set as the first threshold value. Alternatively, the observer may select the range of the obtained image and set the minimum luminance as a threshold value. Furthermore, the observer may select a range of the obtained multiphoton fluorescence image, and the average luminance of pixels in the range may be set as the second threshold value. Further, the highest luminance of the reference image may be set as the second threshold value.

  In the present embodiment, the setting value of the laser intensity adjusting unit 3 such as an acousto-optic element that adjusts the intensity of the laser beam is one of the hardware setting values. Instead, an ultrashort pulse laser light source is used. The number of pulses of the ultrashort pulse laser beam generated from 2 may be set as the hardware set value.

In this embodiment, the setting value of the laser intensity adjusting unit 3 and the applied voltage HV of the photodetector 12 are exemplified as an example of the hardware setting value to be calculated and stored. In addition to this, the scanner 4 The scanning speed SS may be calculated.
In this case, as shown in FIG. 8, before step S29 for increasing the applied voltage HV of the photodetector 12, it is determined whether or not the applied voltage HV of the photodetector 12 is the upper limit (step S32). , if unable to increase any more applied voltage HV, reduce the scanning speed SS of the scanner 4 (step S33), calculates the luminance condition (step S34), the brightness condition to match the brightness condition I ₀ of the reference image Is determined (step S35).

If it is determined that the calculated brightness condition is not identical to the brightness condition I ₀ in the reference image, steps S33~S35 are repeated. On the other hand, in step S35, when the calculated brightness condition becomes identical to the luminance conditions I ₀ in the reference image, step S26 is executed.
By such processing of the control unit 13, for example, hard wafer setting values (setting value (ND) of the laser intensity adjustment unit 3 and setting value (HV) of the photodetector 12, the setting value of the scanner 4), as shown in FIG. Scanning speed (SS) is obtained.

  In this embodiment, as the condensing position of the ultrashort pulse laser beam in the sample A becomes deeper, the laser intensity is first increased until reaching the upper limit, and then the voltage HV applied to the photodetector 12 is increased. It was decided to increase until the upper limit was reached. In the above modification, the scanning speed SS of the scanner 4 is further decreased after the voltage HV applied to the photodetector 12 reaches the upper limit. By doing in this way, generation | occurrence | production of noise can be reduced and a clear multiphoton fluorescence image can be obtained.

  However, the adjustment order of these hardware setting values is not limited to this order, and the applied voltage HV of the photodetector 12 may be increased without increasing the laser intensity. By doing in this way, the damage which the ultrashort pulse laser beam gives to the sample A can be reduced.

  In the present embodiment, the case where the hardware setting values are adjusted one by one has been described, but instead of this, as shown in FIG. 10, two or more hardware setting values (in the example shown in the figure). The laser intensity ND and the voltage HV applied to the photodetector 12 may be adjusted simultaneously.

Further, in the present embodiment, when calculating the luminance condition of the multiphoton fluorescence image taken at each depth position, it is calculated based on the luminance value in one image, but instead, The luminance condition may be calculated based on two or more multiphoton fluorescence images photographed at a minute time interval at each depth position.
For example, the average luminance of pixels having a luminance equal to or higher than a predetermined threshold of two or more multiphoton fluorescence images may be calculated, or the average luminance may be calculated using only a portion having a strong correlation for each pixel. By doing so, it is possible to remove the image noise included only in one multiphoton fluorescence image and calculate the brightness condition with high accuracy.

  Alternatively, the spatial frequency of the multiphoton fluorescence image captured at each depth position is calculated for each pixel, and pixels having a spatial frequency equal to or higher than a predetermined threshold are excluded from the basis for calculating the luminance condition. Also good. Usually, since image noise has a high spatial frequency, the luminance condition can be calculated with high accuracy by effectively excluding it.

  In the present embodiment, the hardware set value is calculated and stored for each depth position, but in this case, it is associated with the type of sample A to be observed and the type of reagent to be used. It may be memorized. In this way, once the type of sample A or the type of reagent is determined, a hardware setting value capable of acquiring a multiphoton fluorescence image suitable for the type of sample or reagent can be quickly called. Further, the hardware setting value may be stored in association with the identification information of the observer. Since the preferred brightness condition differs for each observer, a hardware setting value capable of acquiring a multiphoton fluorescence image that matches the observer's preference can be quickly called up.

  In the present embodiment, the scanning optical device 1 that can irradiate the sample A only with the ultrashort pulse laser beam for excitation is illustrated, but in addition to this, the ultrashort pulse laser beam for light stimulation is used as the sample. You may apply to the scanning optical apparatus which can irradiate A. In this case, the intensity of the ultrashort pulse laser light for light stimulation is calculated based on the intensity of the ultrashort pulse laser light set for each depth of the condensing position of the ultrashort pulse laser light for excitation. It may be. By doing in this way, it becomes possible to perform uniform light stimulation irrespective of the depth of light stimulation.

  In the present embodiment, based on the multiphoton fluorescence images acquired at a plurality of representative depth positions, the hardware setting values at other depth positions are interpolated. Multi-photon fluorescence images at all depth positions to be observed may be acquired, and hardware setting values may be calculated and stored for each. By doing in this way, it becomes possible to acquire a multiphoton fluorescence image with less brightness unevenness for each depth.

In the present embodiment, the hardware setting value is calculated and stored so that the luminance condition for each depth matches the luminance condition I ₀ of the reference image. It is not limited to coincidence, and an error of about several percent may be allowed.

1 is an overall configuration diagram showing a scanning optical device according to an embodiment of the present invention. 2 is a flowchart for explaining processing by a control unit of the scanning optical device in FIG. 1. 3 is a flowchart for explaining luminance condition setting processing in the processing of FIG. 2. 3 is a flowchart for explaining hardware setting value setting processing in the processing of FIG. 2. It is a graph which shows an example of the relationship with the depth of the hardware setting value memorize | stored in the flowchart of FIG. 5 is a graph showing an example of hardware setting values stored for a plurality of depth positions in the flowchart of FIG. 4. It is a graph which shows the hardware setting value after the interpolation process in the flowchart of FIG. 6 is a flowchart showing a modification of processing by the control unit of the scanning optical device in FIG. 1. It is a graph which shows an example of the relationship with the depth of the hardware setting value memorize | stored in the flowchart of FIG. 6 is a graph illustrating an example of a relationship with a depth of a hardware setting value acquired by a modification of the hardware setting value setting process of FIG. 4.

Explanation of symbols

A Sample 1 Scanning optical device 2 Light source for multiphoton excitation 4 Scanner (scanning unit)
12 Photodetector (Fluorescence detector)
13 Control unit (hardware setting adjustment unit, set value storage unit, brightness condition setting unit)
16 Condensing depth adjustment unit

Claims (6)

A light source for multiphoton excitation that emits ultrashort pulse laser light;
A scanning unit that scans the sample with the ultrashort pulse laser beam emitted from the light source for multiphoton excitation;
A condensing depth adjusting unit that adjusts the depth of the condensing position in the sample of the ultrashort pulse laser beam scanned by the scanning unit;
A fluorescence detection unit for detecting fluorescence emitted from a condensing position of the ultrashort pulse laser beam in the sample;
A reference luminance serving as an index from a multiphoton fluorescence image acquired from a predetermined depth position based on the luminance information of the fluorescence detected by the fluorescence detection unit and the scanning position of the ultrashort pulse laser beam by the scanning unit A reference luminance condition setting section for setting conditions;
Wherein a fluorescence detector fluorescence intensity information detected by the luminance condition of the multiphoton fluorescence image is obtained based on the scanning position of the ultrashort pulsed laser light by the scanning unit, the sample of ultra-short pulse laser beam The light collection depth adjusting unit is operated and the light source for multiphoton excitation and the scanning are set so that the reference luminance condition set by the reference luminance condition setting unit is substantially the same for each depth of the condensing position in A hardware setting adjustment unit that adjusts at least one hardware setting value of the fluorescence detection unit ,
The at least one hardware setting value of the light source for multiphoton excitation, the scanning unit, or the fluorescence detection unit in which the luminance conditions of the multiphoton fluorescence image are substantially the same is focused on the ultrashort pulse laser beam in the sample. A hardware setting value storage unit that stores a plurality of depths corresponding to different positions;
A controller that acquires a multiphoton fluorescence image of a sample over a desired depth range using the stored hardware setting value, and
Scanning optical device. Before Symbol hardware settings adjuster unit, the hard wear-set value a plurality of setting values stored in the storage unit by interpolation calculation, the scanning optical apparatus according to claim 1 for adjusting the set value.   The scanning optical device according to claim 1, further comprising a luminance condition setting unit configured to set a luminance condition of the multiphoton fluorescence image based on the acquired image.   The scanning optical device according to claim 3, wherein the luminance condition of the multiphoton fluorescence image is an average luminance value of pixels having luminance equal to or higher than a predetermined threshold value.   The luminance condition of the multiphoton fluorescence image is a number of pixels having a luminance equal to or higher than a first threshold value, and a pixel having a luminance equal to or higher than a second threshold value that is larger than the first threshold value. The scanning optical device according to claim 3, wherein the scanning optical device has a ratio to the number of pixels.   The scanning optical apparatus according to claim 5, wherein the second threshold value is a maximum luminance that can be expressed in the multiphoton fluorescence image.

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