JPH10104079A - Depolarization imaging device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provided a depolarization imaging device with the capability of high correctly specifying a position where a target having a combination of fluorescence probes exists by detecting under a microscope depolarization two-dimensional images of fluorescence generated through the irradiation of excitation light to a specimen. SOLUTION: Pulse excitation light of linear polarization outputted from a pulse excitation light source is allowed to be in a predetermined polarization direction by a 1/2 wavelength plate 2 to then be irradiated on a spacemen 7. Fluorescence emitted from the specimen is permitted to branch in respective linear polarization components of (p)-polarization and (s)-polarization by a polarization beam splitter 10 to subsequently be image-picked up by image intensifiers 13, 14. Anisotropic two-dimensional images of fluorescence is obtained by being corrected in its polarization response based on the picked up images.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a depolarization imaging technique for detecting the depolarization of fluorescence generated when a fluorescently labeled sample is irradiated with excitation light.

[0002]

2. Description of the Related Art When a fluorescently labeled sample is irradiated with linearly polarized excitation light, fluorescence is generated from the fluorescent probe. The polarization state of the fluorescence is linearly polarized at the beginning of the excitation light irradiation.However, when the fluorescent probe rotates irregularly due to Brownian motion, the molecular axis of the excited fluorescent probe is disturbed. The polarization state of the fluorescence is cancelled, and finally the fluorescence state becomes unpolarized. If a two-dimensional image of the degree of fluorescence depolarization can be detected, it is possible to specify the position where the target to which the fluorescent probe is bound is present, and to analyze the behavior of the target in the sample. Therefore, it is expected to contribute to elucidation of various functions in cells, for example. However, an apparatus for detecting a two-dimensional image of depolarized fluorescence at a time with a two-dimensional detector having a time resolution and specifying a position where a target to which a fluorescent probe is bonded is present has not yet been known.

By the way, conventionally, there is known a technique for detecting a two-dimensional image of the intensity and polarization state of fluorescence generated by irradiating a fluorescently labeled sample with excitation light.

First, there is a time-resolved fluorescence microscope as a method for detecting a two-dimensional image of fluorescence intensity with high sensitivity. This time-resolved fluorescence microscope uses the fact that the fluorescence lifetime of autofluorescence (background light) generated from a sample or an optical system is different from the fluorescence lifetime of fluorescence (signal light) generated from a fluorescent probe, and thus the S / B ratio is improved. (Signal light intensity / background light intensity). For example, as shown in FIG. 27, by using a fluorescent probe having a fluorescence lifetime longer than the fluorescence lifetime of auto-fluorescence generated from a sample or an optical system, and measuring the fluorescence intensity when the auto-fluorescence is sufficiently attenuated, Only the intensity of the fluorescence generated from the probe is measured.

A technique for observing a two-dimensional image of the polarization state of fluorescence is disclosed in D. Axelrod, "Carbocyanine Dye Orientation.
in Red Cell Membrane studied by Microscopic Fluor
escence Polarization ", Biophys.J., Vol.26, pp.557-
574 (1979) and K. Suzuki, et al, "Spatiotemporal
Relationships Among Early Events of Fertilization
in Sea Urchin Eggs Revealed by Multiview Microsco
py ", Biophys. J., Vol. 68, pp. 739-748 (1995).

[0006] The technique described in D. Axelrod's paper is based on the measurement of the polarization state of fluorescence generated by the irradiation of stationary light (polarized excitation light) under a microscope, whereby the fluorescent dye introduced into a biological membrane is measured. It examines orientation and mobility. The photodetector used here is a photomultiplier tube, and obtains a two-dimensional fluorescence polarization image by scanning a stop on a fluorescence image plane. In measuring the polarization of such fluorescence, measurement errors may occur due to different responses of the optical system and the photodetector to different polarization orientations. The measurement error is corrected (polarization response correction) by passing the light through a light receiving optical system (optical system from the sample to the photodetector) and the photodetector.

The technology described in the paper of K. Suzuki is:
Analyzing the steady-state molecular orientation of the sample by splitting the fluorescence generated from the sample into linearly polarized light components orthogonal to each other by a polarizing beam splitter, and capturing each of the two split images of the fluorescence with a single camera Is what you do. In the technique described in this paper, a homogeneous sample is excited with non-polarized excitation light, and polarization response correction is performed using the fact that the fluorescence generated from the sample is completely unpolarized.

[0008]

However, the time-resolved fluorescence microscope described above is effective for separating the signal light from the background light, but detects the polarization state of the separated signal light. Is not performed, so that depolarization cannot be detected. Therefore, it is impossible to detect whether the fluorescence is generated from the fluorescent probe bound to the target or from the free fluorescent probe.

In the technique described in D. Axelrod's paper and K. Suzuki's paper, although the polarization state of fluorescence can be detected two-dimensionally, it is described in K. Suzuki's paper. The technique cannot detect the degree of depolarization of the fluorescence, and therefore cannot distinguish between the fluorescence generated from the fluorescent probe bound to the target and the fluorescence generated from the free fluorescent probe. Also, D.Ax
The technique described in elrod's paper has low sensitivity to detect depolarization due to the use of stationary light as excitation light, and therefore, fluorescence generated from the fluorescent probe bound to the target and fluorescent light generated from the free fluorescent probe. And the ability to identify

Further, in the techniques described in the papers of D. Axelrod and K. Suzuki, polarization response correction is performed using unpolarized light, but this unpolarized light should be observed originally. Correction accuracy is low because it is different from fluorescence. Also, it is difficult to create ideal non-polarized light.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-described problem, and it is possible to detect a two-dimensional image of depolarized fluorescence generated by irradiating a fluorescently labeled sample with excitation light under a microscope. It is another object of the present invention to provide a depolarization imaging apparatus capable of specifying, with high accuracy, a position where a target to which a fluorescent probe is bound is present.

[0012]

According to the present invention, there is provided a depolarization imaging apparatus comprising: (1) a pulse excitation light source for outputting a linearly polarized pulse excitation light for exciting a sample; and (2) an output from the pulse excitation light source. (3) an irradiation optical system for irradiating the sample from a predetermined direction with the pulse excitation light output from the polarization azimuth rotation means, and (4) a pulse excitation light. Inputs the fluorescence generated by irradiating the sample from a direction substantially the same as a predetermined direction, branches into linear polarization components of first and second polarization directions orthogonal to each other, and separates first and second images from each other. (5) first imaging means for imaging a linearly polarized light component of the first polarization direction of the fluorescence imaged on the first imaging plane; Line of the second polarization direction of the fluorescence imaged on the image plane No. 2 A second imaging means for imaging the light component; (7) a control means for controlling the imaging timing of each of the first and second imaging means; and (8) an image taken by each of the first and second imaging means. Image calculation means for obtaining a two-dimensional image of depolarization of the fluorescence generated from the sample based on the images of the linear polarization components of the first and second polarization directions of the fluorescence.

According to the depolarized imaging apparatus,
The linearly polarized pulse excitation light for exciting the sample is output from the pulse excitation light source unit, the polarization direction is rotated by the polarization direction rotating means to a predetermined polarization direction, and the sample is irradiated from the predetermined direction by the irradiation optical system. . Fluorescence generated by irradiating the sample with the pulsed excitation light is input by the light receiving optical system from a direction substantially the same as a predetermined direction, and is linearly intersected at first and second polarization directions (p-polarized light and s-polarized light). The first and second light beams are split into polarized light components and different from each other.
Are imaged on each of the image forming planes, and the image is taken by each of the first and second image pickup means whose image pickup timing is controlled by the control means. Then, the two-dimensional image of the depolarization of the fluorescence generated from the sample is converted by the image calculation means into the linear polarization components of the first and second polarization directions of the fluorescence imaged by the first and second imaging means, respectively. Determined based on the image.

In this case, the calculating means includes an image of the linearly polarized light component of each of the first and second polarization directions of the fluorescence imaged when the sample is irradiated with the pulsed excitation light having the first polarization direction; Further, the polarization response correction is performed based on the images of the linearly polarized light components of the first and second polarization directions of the fluorescence imaged when the sample is irradiated with the pulsed excitation light having the second polarization direction. To obtain a two-dimensional image of the depolarization of the fluorescence. By doing this,
When the molecular axes of the fluorescent probes in the sample are not oriented and are distributed at random, the polarization response can be corrected appropriately, and a two-dimensional image of the depolarized fluorescence can be obtained with high accuracy. .

[0015] The calculating means may be configured to irradiate the sample located at a predetermined rotation angle position about a predetermined direction with a pulsed excitation light having a first polarization direction to a first rotation of the fluorescence. The image of the linearly polarized light component in each of the second polarization direction and the predetermined rotation angle position with respect to the central axis is 9
When the sample at the rotation angle position different by 0 degrees is irradiated with the pulsed excitation light having the second polarization direction, the images of the linearly polarized light components of the first and second polarization directions of the fluorescence imaged are obtained. Based on this, a two-dimensional image of the depolarization of fluorescence may be obtained by performing polarization response correction. In this way, when the molecular axes of the fluorescent probes in the sample are oriented and distributed, the polarization response can be corrected appropriately, and a two-dimensional image of the depolarization of the fluorescence can be obtained with high accuracy. Can be.

[0016]

Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

First, prior to the description of the configuration of the depolarization imaging apparatus according to the present invention, the fluorescence depolarization method employed by the depolarization imaging apparatus according to the present invention will be described. Fluorescence depolarization is a method of using a fluorescent probe or a target (for example, a protein) labeled with the fluorescent probe.
It is an effective means for knowing the exercise of a person. That is, if the fluorescent probe rotates irregularly due to Brownian motion during the excitation lifetime until the fluorescent probe excited by the excitation light generates fluorescence, the molecular axis of the excited fluorescent probe is disturbed, and the generated fluorescent light is disturbed. Is eliminated.

The degree of the fluorescence depolarization is determined by the speed of the Brownian motion of the fluorescent probe or the target labeled by the fluorescent probe.
As a result, the movement of the fluorescent probe or the target labeled with the fluorescent probe can be known. That is, a free fluorescent probe performs Brownian motion faster than a fluorescent probe bound to a target, so that depolarization is faster. Therefore, the fluorescence depolarization method makes it possible to distinguish between a fluorescent probe bound to a target and a free fluorescent probe.

Based on the concept of the fluorescence depolarization method,
It is conceivable to determine whether or not the fluorescent probe is bound to the target by measuring the depolarization of the fluorescence generated by the excitation of the constant light (CW light). However, the number of free fluorescent probes bound to the target can be considered. If the number is extremely large compared to the number of fluorescent probes (this is a very common case), the detection sensitivity is very poor.

Therefore, in the depolarization imaging apparatus according to the present invention, as shown in FIG. 1, the pulse excitation light is irradiated by utilizing the fact that the free fluorescent probe depolarizes faster than the fluorescent probe bound to the target. The number of free fluorescent probes is overwhelmingly larger than the number of fluorescent probes bound to the target by removing the fast depolarized component of the generated fluorescence by the time gate method and imaging with the photodetector. Even in this case, the position in the sample where the fluorescent probe bound to the target exists is detected.

Next, the polarization response correction employed by the depolarization imaging apparatus according to the present invention will be described. One of the major factors in the measurement error in the depolarization measurement is the polarization characteristics of the light receiving optical system (the optical system from the sample to the photodetector) and the photodetector. Ideally, the receiving optics and photodetector should respond equally to different polarizations, but in practice respond differently depending on the polarization direction. Therefore, to determine the polarization of true fluorescence,
The polarization response needs to be corrected.

This correction amount of the polarization response is usually called a G factor, and is defined as follows in a conventional zero-dimensional (point) depolarization measuring apparatus. FIG. 2 is an explanatory diagram of how to obtain a G factor in a conventional depolarization measuring device. Linearly polarized excitation light polarized in the p-direction is incident on the sample to generate fluorescence. Fluorescence of linearly polarized light components in each of the p direction (direction parallel to the p direction of the excitation light) and the s direction (direction perpendicular to the p direction of the excitation light) using a polarizer from a direction orthogonal to the incident direction. Are measured, and their intensities are _defined as I _pp and I _ps . Then, as an amount characterizing the degree of polarization of fluorescence, the fluorescence anisotropy ratio r is

(Equation 1) Is defined in

However, since the actually measured fluorescence intensity is obtained through the light receiving optical system and the photodetector, it reflects the anisotropy thereof. That is, assuming that the response of the light receiving optical system and the photodetector to p-polarized light is T _p and the response to s-polarized light is T _s , the actual measured fluorescence intensities I _pp ′ and I _ps ′ are the true fluorescence intensities, respectively. Between I _pp and I _ps respectively

(Equation 2) There is a relationship. Therefore, the relationship between the fluorescence anisotropy ratio r and the measured fluorescence intensities I _pp ′, I _ps ′ is obtained by substituting equation (2) into equation (1).

(Equation 3) It is expressed by the following relational expression. The depolarization of the fluorescence can be determined from the value of the fluorescence anisotropy ratio r.

Incidentally, the ratio of the response T _p to _p- polarized light and the response T _s to s-polarized light, which appears in equation (3),

(Equation 4) Is a correction amount called a G factor. This G factor is obtained as follows. The sample is irradiated with s-polarized excitation light, and the fluorescence of the linearly polarized light components of the p-polarized light and s-polarized light of the generated fluorescence is measured. The respective intensities are defined as I _sp ′ and I _ss ′. Between each of the intensities I _sp and I _ss , as in equation (2),

(Equation 5) There is a relationship. Since it is considered that the intensity I _sp of the p-polarized fluorescence and the intensity I _ss of the s-polarized fluorescence are equal to each other with respect to the s-polarized excitation light, from the equation (5),

(Equation 6) Therefore, the G factor represented by the equation (4) is
Using the measured fluorescence intensities I _sp ′ and I _ss ′,

(Equation 7) Is required. That is, the depolarization of the fluorescence can be obtained with high accuracy by performing the polarization response correction by the G factor on the fluorescence anisotropy ratio r.

The polarization response correction described above is for a conventional zero-dimensional (point) depolarization measuring apparatus. In the depolarization imaging apparatus according to the present invention, the sample is irradiated with excitation light. Since the direction and the direction of observing the fluorescence are the same, the polarization response correction is performed as follows. FIG. 3 is an explanatory diagram of how to obtain the G factor in the depolarization imaging apparatus according to the present invention.

The p-polarized excitation light is reflected by the dichroic mirror and is incident on the sample to generate fluorescence, and the intensities I _pp and I p of the p-polarized light and the s-polarized light of the fluorescence generated by the sample and transmitted through the dichroic mirror, respectively. Measure _ps . The fluorescence anisotropy ratio r is

(Equation 8) It is represented by

However, even in the case of the depolarization imaging apparatus according to the present invention, the actually measured fluorescence intensity is obtained through the light receiving optical system and the photodetector, and therefore reflects the anisotropy thereof. . That is, assuming that the response of the light receiving optical system and the photodetector to p-polarized light is T _p and the response to s-polarized light is T _s , the actual measured fluorescence intensities I _pp ′ and I _ps ′ are the true fluorescence intensities, respectively. Between I _pp and I _ps respectively

(Equation 9) There is a relationship. Therefore, the relationship between the fluorescence anisotropy ratio r and the measured fluorescence intensities I _pp ′, I _ps ′ is

(Equation 10) The depolarization of the fluorescence can be obtained from the value of the fluorescence anisotropy ratio r. Here, G factor is

[Equation 11] It is represented by

As a method of obtaining the G factor, a sample is excited by excitation light of linearly polarized light having a direction parallel to the fluorescence imaging direction from a direction perpendicular to the fluorescence imaging direction. A method of obtaining the same is also conceivable. However, it is difficult for the excitation light to be incident on the sample from such a direction because of restrictions of a sample cell, a sample stage, and the like. Therefore, in the depolarization imaging apparatus according to the present invention, the G factor is obtained as follows. In the following, the factor G is determined in two ways: the case where the molecular axes of the fluorescent probes in the sample are not oriented and are distributed at random, and the case where the molecular axes of the fluorescent probes in the sample are oriented and distributed. Are described separately.

First, a method of obtaining the G factor when the molecular axes of the fluorescent probes in the sample are not oriented and are distributed at random will be described.

The intensities (true values) of the p-polarized component and the s-polarized component of the fluorescence generated by the irradiation of the sample with the p-polarized excitation light are I _pp and I _ps , and the measured values are I _pp ′ and I _ps ′. . Also, the intensities (true values) of the p-polarized component and the s-polarized component of the fluorescence generated by irradiating the sample with the s-polarized excitation light are I _sp and I _ss , and the measured values are I _sp ′ and I _ss ′. At this time, these quantities, the response T _p to the p-polarized light of the light receiving optical system and the photodetector, and the response T _s to the s-polarized light
In between

(Equation 12)

(Equation 13) There is a relationship.

Further, the fluorescence anisotropy ratio r _p obtained by irradiation with p-polarized excitation light is

[Equation 14] The fluorescence anisotropy ratio r _s obtained by irradiation with s-polarized excitation light is represented by the following formula:

(Equation 15) It is represented by the following formula. G factor is

(Equation 16) Equations (14) and (15) can be expressed as

[Equation 17]

(Equation 18) It is represented by

[0032] When the molecular axis of the fluorescent probes in the sample is completely randomly distributed is the fluorescence anisotropy ratio r _p obtained for irradiation of the p-polarized light of the excitation light, the irradiation of the s-polarized light of the excitation light The resulting fluorescence anisotropy ratio r _s is equal,

[Equation 19] Becomes By substituting the expressions (17) and (18) into the expression (19), the G factor becomes

(Equation 20) Will be given by That is, when the molecular axes of the fluorescent probes in the sample are not oriented and are distributed at random, the G factor is the p-polarized light intensity and the p-polarized light intensity of the fluorescence generated by the sample irradiation of the p-polarized light and the s-polarized light. The s-polarized light intensity is measured, and based on these intensities, it can be determined by equation (20). The depolarization of the fluorescence can be determined with high precision by performing the polarization response correction by factor G obtained in this way for fluorescence anisotropy ratio r _p and r _s.

Next, a method of obtaining the G factor when the molecular axes of the fluorescent probes in the sample are oriented and distributed will be described. Note that an actual measurement target such as a cell membrane is often close to such a system.

In this case, if the above-described method of determining the G factor when the molecular axes of the fluorescent probes in the sample are not oriented and are distributed at random is applied, the light receiving optical system and the photodetector become polarized. Even if it does not have anisotropy, the fluorescence is measured as if it had anisotropy. Therefore, when the molecular axes of the fluorescent probes in the sample are oriented and distributed, the assumption of equation (19) does not hold, and the fluorescence anisotropy ratio r _p obtained for irradiation of p-polarized excitation light is , Is generally different from the fluorescence anisotropy ratio r _s obtained for irradiation with s-polarized excitation light,

(Equation 21) It is.

That is, the intensity of each of the p-polarized component and the s-polarized component of the fluorescence generated from the sample by the irradiation of the p-polarized excitation light is defined as I _pp and I _ps , and the intensity of the fluorescence generated from the sample by the irradiation of the s-polarized excitation light. _Assuming that the intensities of the p-polarized component and the s-polarized component of the fluorescent light are _Isp and _Isss , respectively, when the molecular axes of the fluorescent probes in the sample are completely randomly distributed,

(Equation 22) However, when the molecular axis of the fluorescent probe is oriented and distributed,

(Equation 23) It is.

Therefore, when the molecular axis of the fluorescent probe is oriented and distributed, the factor G is obtained as follows. That is, the intensity (true value) of each of the p-polarized component and the s-polarized component of the fluorescence generated from the sample by the irradiation of the s-polarized excitation light is defined as I _sp (0) and I _ss (0), and the measured value is defined as I _sp (0). _sp (0) 'and I _ss (0)'. Further, as shown in FIG. 4, after rotating the sample by 90 degrees with the excitation light irradiation direction as the central axis, each of the p-polarized component and the s-polarized component of the fluorescence generated from the sample by the irradiation of the p-polarized excitation light. The intensities (true values) are I _pp (90) and I _ps (90), and the measured values are I _pp (90) ′ and I _ps (90) ′. At this time,
Between these quantities, the response T _{p of} the receiving optical system and the photodetector to p-polarized light, and the response T _s to s-polarized light,

(Equation 24)

(Equation 25) There is a relationship.

The fluorescence anisotropy ratio r _s (0) before rotating the sample is

(Equation 26) Where the fluorescence anisotropy ratio r _p (90) after rotating the sample by 90 degrees is

[Equation 27] It is represented by the following formula. Here, using the G factor represented by the equation (16), the equations (26) and (27) are respectively

[Equation 28]

(Equation 29) It is represented by

When the molecular axis of the fluorescent probe in the sample is oriented and distributed, the fluorescence obtained by the irradiation of the p-polarized excitation light after rotating the sample by 90 degrees around the excitation light irradiation direction as the central axis. The anisotropy ratio r _p (90) is equal to the fluorescence anisotropy ratio r _s (0) obtained for s-polarized excitation light irradiation before rotating the sample. That is,

[Equation 30] It is. Therefore, Equations (28) and (29) are replaced by (3
0)

(Equation 31) Is obtained, and this is solved, and as an expression representing the G factor,

(Equation 32) Is obtained. That is, when the molecular axis of the fluorescent probe in the sample is oriented and distributed, the G factor is obtained by irradiating the sample with the excitation light of each of the p-polarized light and the s-polarized light with respect to the rotation angle of the sample with the excitation light irradiation direction as the central axis. The intensity of the p-polarized light and the intensity of the s-polarized light of the fluorescent light generated by irradiating the sample with the excitation light of the p-polarized light and the s-polarized light are measured based on these intensities.
It is obtained by the formula. The depolarization of the fluorescence can be obtained with high accuracy by correcting the polarization response by the G factor obtained in this way for the fluorescence anisotropy ratios r _p (90) and r _s (0).

Next, the configuration of the depolarization imaging apparatus according to the present invention will be described. FIG. 5 is a configuration diagram of the depolarization imaging apparatus according to the present invention.

The pulse excitation light source 1 outputs pulse excitation light for exciting a fluorescent probe in the sample 7. The pulsed excitation light output from the pulsed excitation light source 1 is converted into linearly polarized light by the polarizer 2, and then enters a half-wave plate (polarization direction rotating means) 3. Rotated to any of the polarization orientations. The sample 7 is irradiated with the p-polarized or s-polarized pulsed excitation light by the irradiation optical system. That is, the pulsed excitation light has a predetermined light beam diameter and shape by the excitation light introducing optical system 4, is reflected by the dichroic mirror 5, is condensed by the objective lens 6, and is on the sample 7 placed on the sample stage 8. The observation area is irradiated. The sample stage 8 can rotate the sample 7 about the optical axis of the pulsed excitation light incident on the sample 7.

When the sample 7 is irradiated with the pulse excitation light, fluorescence is generated from the fluorescent probe in the sample 7. The fluorescence emitted by the light receiving optical system in the same direction as the excitation light irradiation direction is separated into p-polarized light and s-polarized light to form an image. That is, of the fluorescent light emitted from the sample 7, the light beam incident on the objective lens 6 passes through the objective lens 6, passes through the dichroic mirror 5, passes through the band-pass filter 9, and is converted into a p-polarized component by the polarizing beam splitter 10. The s-polarized light component is divided into two, and the p-polarized light component and the s-polarized light component are respectively polarized by the imaging lenses 11 and 12, respectively. (Imaging plane).

Here, the band-pass filter 9 transmits the fluorescent light, but absorbs the scattered light of the pulse excitation light generated in the sample 7, and the polarizing beam splitter 10 controls the p-polarized light component of the fluorescent light. Is transmitted, and the s-polarized light component is reflected. Each of the image intensifiers 13 and 14 with a gate function opens and closes the gate based on the gate signal output from the gate controller (control means) 15, and the fluorescence polarization imaged on the imaging surface when the gate is open This is for capturing an image. Also,
The gate controller 15 instructs the timing of the pulsed pump light is output from the pulsed excitation light source 1 as a reference time, the time t ₀ from the time Δt image intensifier 13 and 14 with only the gate function between to open the respective gate Outputs a gate signal.

The fluorescence polarization images of the p-polarized light component and the s-polarized light component captured by the image intensifiers 13 and 14 with the gate function, respectively, are input to the image calculation unit 16. The image calculation unit 16 obtains a two-dimensional image of the fluorescence anisotropy ratio based on these fluorescence polarization images, and further performs polarization response correction.

Next, a method for obtaining a two-dimensional image of the fluorescence anisotropy ratio corrected for the polarization response using the depolarization imaging apparatus will be described. The two-dimensional image of the polarization response correction and the fluorescence anisotropy ratio is obtained by calculation in the image calculation unit 16. In the following,
The case where the molecular axes of the fluorescent probes in the sample 7 are not oriented and are randomly distributed and the case where the molecular axes of the fluorescent probes in the sample 7 are oriented and distributed will be described separately.

If the molecular axes of the fluorescent probes in the sample 7 are not oriented and are distributed at random, a two-dimensional image of depolarized light is obtained as follows.

In this case, the optical axis of the half-wave plate 3 is appropriately set, and the sample 7 is irradiated with the pulse excitation light output from the pulse excitation light source 1 as p-polarized light. In this case, the instruction of the gate signal from the gate controller 15, the p-polarized light and s polarized light, respectively fluorescence image captured by the image intensifier 13 and 14 respectively with gate function during the period from the time t ₀ time Delta] t, I _pp (t ₀ ,
Δt) and I _ps (t ₀ , Δt). This time t ₀ is
This is a time after the polarization of the fluorescence generated from the free fluorescent probe is sufficiently resolved and the fluorescence generated from the fluorescent probe bound to the target has a measurable intensity.

Similarly, the optical axis of the half-wave plate 3 is appropriately set, and the sample 7 is irradiated with the pulse excitation light output from the pulse excitation light source 1 as s-polarized light. At this time, according to the instruction of the gate signal from the gate controller 15,
Image intensifiers 13 and 1 with gate function
4 p-polarized light and s polarized light, respectively fluorescence images captured by the _{respective, I sp (t 0, Δt} ) and I _ss (t _0, Δ
t).

Then, the G factor relating to the polarization response correction is

[Equation 33] And the fluorescence anisotropy ratio r (t ₀ , Δt) is calculated as

(Equation 34) Ask for. Note that the fluorescence images I _pp (t ₀ , Δt), I
_ps (t ₀ , Δt), I _sp (t ₀ , Δt) and I _ss (t ₀ , Δ
t), G factor and fluorescence anisotropy ratio r (t ₀ , Δt)
All are functions of coordinates (x, y) on a plane perpendicular to the optical axis of the pulsed excitation light applied to the sample 7. In this way,
A two-dimensional depolarized image of only the fluorescence generated from the fluorescent probe bound to the target can be detected with high accuracy.

When the molecular axes of the fluorescent probes in the sample 7 are oriented and distributed, a two-dimensional depolarized image is obtained as follows.

In this case, the sample 7 is irradiated with the pulse excitation light output from the pulse excitation light source 1 as s-polarized light by appropriately setting the optical axis of the half-wave plate 3. In this case, the instruction of the gate signal from the gate controller 15, the p-polarized light and s polarized light, respectively fluorescence image captured by the image intensifier 13 and 14 respectively with gate function during the period from the time t ₀ time Delta] t, I _sp (0
°, t _0, Δt) and I _{s s} (0 °, t _0, Δt) to.
The time t ₀ is a time after the polarization of the fluorescence generated from the free fluorescent probe has been sufficiently eliminated and the fluorescence generated from the fluorescent probe bound to the target has a measurable intensity.

Subsequently, after rotating the sample 7 by 90 degrees by rotating the sample stage 8, the half-wave plate 3
Is set appropriately, and the sample 7 is irradiated with the pulsed excitation light output from the pulsed excitation light source 1 as p-polarized light.
At this time, according to the instruction of the gate signal from the gate controller 15, the fluorescence images of the p-polarized light and the s-polarized light captured by the image intensifiers 13 and 14 with the gate function are respectively converted into I _pp (90 °, t ₀ , Δ).
t) and I _ps (90 °, t ₀ , Δt).

Then, the G factor relating to the polarization response correction is

(Equation 35) And the fluorescence anisotropy ratio r _s (t ₀ , Δt) when the sample is irradiated with the s-polarized excitation light,

[Equation 36] Ask for. Note that the fluorescence images I _sp (0 ゜, t ₀ , Δt), I
_ss (0 °, t ₀ , Δt), I _pp (90 °, t ₀ , Δt) and I _ps (90 °, t ₀ , Δt), G factor, and fluorescence anisotropy ratio r _s (t ₀ , Δt) ) Are all functions of coordinates (x, y) on a plane perpendicular to the optical axis of the pulsed excitation light applied to the sample 7. In this manner, a two-dimensional image of only the fluorescence generated from the fluorescent probe bound to the target can be detected with high accuracy.

Next, a description will be given of the results of an experiment performed on polarization response correction in the depolarization imaging apparatus according to the present invention. The specific configuration of the depolarized imaging apparatus used here is as follows. The pulse excitation light source 1 is a mode-locked titanium sapphire laser light source, and the pulse excitation light applied to the sample 7 is a second harmonic (wavelength 400 nm, pulse width 2
00 femtoseconds and a repetition frequency of 100 kHz). The polarizer 2 is a Glan laser prism,
The wave plate 3 is a Fresnel rhomb 1/2 wave plate. In addition,
Image intensifiers 13 and 1 with gate function
Here, instead of each of the above, fluorescence intensity was measured by a time-correlated photon counting method (TCPC method) using a photomultiplier tube with a built-in microchannel plate. As sample 7, 17-mer DNA labeled with 5 ′ FITC was used. In this sample 7, the molecular axes of the fluorescent probes are not oriented and are distributed at random. Therefore,
Factor G is determined by equation (20), and fluorescence anisotropy ratio r is determined by equation (17).
It was found by the formula.

FIG. 6 is a graph showing an attenuation curve (thick line) when the intensity of the fluorescence generated from the sample is measured without polarization, and a response function (thin line) of the apparatus under a microscope.
FIG. 7 shows the sample 7 when excited with p-polarized pulsed excitation light.
5 is a graph showing the respective attenuation curves of the p-polarized light component (thick line) and the s-polarized light component (thin line) of the fluorescence generated from FIG. In FIG. 7, the attenuation curve of the s-polarized light component (fine line) is
It is displayed as a value obtained by multiplying the actually measured value by the G factor.

FIG. 8 shows the decay curve of the fluorescence anisotropy ratio obtained by performing the polarization response correction using the factor G (thick line), and the decay curve of the fluorescence anisotropy ratio obtained without performing the polarization response correction. (Thin line). That is, the bold line represents p
The G factor is determined based on the result of the polarization measurement of the fluorescence generated from the sample 7 when excited with the pulsed excitation light of the polarized light and the s-polarized light, and the fluorescence generated from the sample 7 when excited with the pulsed excitation light of the p-polarization. 4 shows the decay curve of the fluorescence anisotropy ratio obtained by performing the polarization response correction by the G factor for the measured values of the intensities of the p-polarized component and the s-polarized component of FIG. On the other hand, the thin line shows the decay curve of the fluorescence anisotropy ratio obtained without performing the polarization response correction by the G factor under the assumption that the actually measured value is a true value. The value of the G factor is
1.076. In this device, it is considered that the dichroic mirror mainly contributes to the G factor.

FIG. 9 shows the decay curve (thick line) of the fluorescence anisotropy ratio obtained by performing polarization response correction under a microscope (the configuration of FIG. 3) such as a depolarization imaging apparatus according to the present invention, and 3 is a graph showing a fluorescence anisotropy ratio decay curve (thin line) obtained by performing polarization response correction in the depolarization measuring device having the configuration (the configuration in FIG. 2). As shown in this figure, both show good agreement. That is, it was confirmed that accurate fluorescence anisotropy ratio can be measured by performing polarization response correction even under a microscope such as a depolarization imaging apparatus according to the present invention.

Next, the results of measurement of fluorescence depolarization using the depolarization imaging apparatus according to the present invention will be described. The specific configuration of the depolarized imaging apparatus used here is as follows. The pulse excitation light source 1 is a mode-locked titanium sapphire laser light source, and the second harmonic (wavelength: 400 nm, pulse width: 200 femtoseconds, repetition frequency: 100 kHz) outputted from the sample 7 is used as pulse excitation light. Using. Polarizer 2
Is a Glan laser prism, and the half-wave plate 3 is
This is a Fresnel rhomb half-wave plate.

Each of the image intensifiers 13 and 14 with a gate function was subjected to analog measurement at a repetition frequency of 2 kHz by thinning out a 100 kHz trigger signal. The gate of each of the image intensifiers 13 and 14 having a gate function has an intensity at which the fluorescence generated from the fluorescent probe bound to the target can be measured after the polarization of the fluorescence generated from the free fluorescent probe is sufficiently eliminated. A fixed time Δ from time t ₀ such that
The gate controller 15 controlled the opening by t.

Sample 7 is an NG cell (nerve-like cell), and the fluorescent probe is HDA that specifically binds to the cell membrane.
F (5- (N-hexadecanoyl) aminofluorescein), the staining concentration was 10 μM, and the staining time was 30 minutes.
In this sample 7, the molecular axes of the fluorescent probes are not oriented and are distributed at random. Therefore, the G factor was determined by equation (20), and the fluorescence anisotropy ratio r was determined by equation (17).

FIG. 10 shows the respective decay curves of the p-polarized component (thick line) and the s-polarized component (thin line) of the fluorescence generated when the free fluorescent probe (HDAF solution) was excited by the p-polarized pulsed excitation light. It is a graph. In this figure, the attenuation curve of the s-polarized light component (thin line) is represented by G
It is displayed as a value multiplied by a factor. FIG. 11 shows a fluorescent probe (H) bound to a target with p-polarized pulsed excitation light.
It is a graph which shows the attenuation curve of each of the p-polarized component (thick line) and the s-polarized component (fine line) of the fluorescence generated when exciting DAF-stained NG cells). In this figure, s
The attenuation curve of the polarization component (thin line) is indicated by a value obtained by multiplying the actually measured value by the G factor.

FIG. 12 shows the HDAF solution (thin line) and H
It is a graph which shows the decay curve of the fluorescence anisotropy ratio obtained by performing the polarization response correction by G factor about the fluorescence generated when exciting DAF-stained NG cells (thick line). That is, the thin line is a decay curve of the fluorescence anisotropy ratio when the fluorescent probe is in a free state, and the thick line is the decay of the fluorescence anisotropy ratio when the fluorescent probe is bound to the target. It is a curve.

FIG. 13 is a graph showing the results of simulation calculation of the fluorescence anisotropy ratio r by the time gate method based on the measurement results shown in FIG. The thin line is the decay curve of the fluorescence anisotropy ratio when the fluorescent probe is in a free state (HDAF solution), and the thick line is when the fluorescent probe is bound to the target (HDAF-stained NG cells). 5 is a decay curve of the fluorescence anisotropy ratio of FIG. In this simulation, in order to obtain a sufficient amount of received light, FIG.
The measured values shown in FIG. 2 were integrated from time t ₀ to infinite time (the time when the fluorescence was sufficiently attenuated), and the time t ₀ was used as a parameter to show a graph.

FIG. 14 shows a free fluorescent probe (HDA).
F solution) and a fluorescent probe (HDA) bound to the target
The resolution in the depolarization measurement between F-stained NG cells) was defined by the contrast value of the fluorescence anisotropy ratio r.
6 is a graph showing the result of obtaining this contrast value based on the simulation result shown in FIG. As can be seen from this graph, the later the gate-on time t ₀ , the better the resolution. This is because the polarization of the fluorescence generated from the free fluorescent probe (HDAF solution) is resolved faster, so that the depolarization measurement is performed after the polarization of the fluorescence from the fluorescent probe has been sufficiently resolved, so that the free fluorescence can be measured. This shows that sufficient resolving power can be obtained between the probe and the fluorescent probe bound to the target.

Next, an imaging photograph of fluorescence depolarization using the depolarization imaging apparatus according to the present invention is shown in FIG.
25 to FIG. The specific configuration of the depolarization imaging apparatus used here is the same as described above.
The same applies to the fluorescent probe.

FIG. 15 is a photograph of a halftone image in which a non-polarized light image of fluorescence generated from a fluorescent probe (HDAF-stained NG cell) bound to a target is displayed on a display. When acquiring this fluorescent image, the gate ON time t ₀ of the image intensifier with a gate function is set to 1
ns, and the time Δt during which the gate is open was 13 ns.

FIGS. 16 to 18 show images of the fluorescence anisotropy ratio of the fluorescence generated from each of the free fluorescent probe (HDAF solution) and the fluorescent probe bound to the target (HDAF-stained NG cells). It is a photograph of the displayed halftone image. The gate ON time t ₀ is ₀ ns in FIG.
Is 1 ns, and in FIG. 18, it is 2 ns. The gate time width Δt is 13 ns in each of FIGS. In each figure, the fluorescence anisotropy ratio imaging of a free fluorescent probe (HDAF solution) is shown on the right, and the fluorescence anisotropy ratio imaging of a fluorescent probe (HDAF-stained NG cells) bound to the target is shown on the left.

FIG. 19 shows the average value and the standard deviation of the fluorescence anisotropy ratio based on the fluorescence anisotropy ratio imaging shown in FIGS. 16 to 18, respectively. 9 is a table showing the results of a simulation calculation of the separation power (contrast value) in depolarization measurement when coexistence is present. As can be seen from this chart, a free fluorescent probe (HDA
Since the polarization of the fluorescence generated from the (F solution) is eliminated earlier, the resolution (contrast value) is improved as the gate ON time t ₀ is delayed.

FIG. 20 shows the HD immersed in the HDAF staining solution.
5 is a photograph of a halftone image in which a phase contrast image of AF-stained NG cells is displayed on a display. FIG. 21 is a photograph of a halftone image in which a non-polarized fluorescent image of HDAF-stained NG cells immersed in an HDAF staining solution is displayed on a display. The gate ON time t ₀ is 2 ns, and the gate time width Δt is about 46 ns.

FIGS. 22 and 23 show HDAF stained N immersed in HDAF stain when excited by steady light.
It is the photograph of the halftone image which displayed imaging of the fluorescence anisotropy ratio of G cell on the display. In FIG. 22, the polarization response correction is performed, but in FIG. 23, the correction is not performed.

FIG. 24 and FIG. 25 are photographs of halftone images showing on a display the imaging of the fluorescence anisotropy ratio of HDAF-stained NG cells immersed in the HDAF staining solution in the case of the time gate method. The gate ON time t ₀ is 2 ns, and the gate time width Δt is about 46 ns. In FIG. 24, the polarization response correction is performed, but in FIG. 25, the correction is not performed.

FIG. 26 shows a free fluorescent probe (HDAF solution) and a fluorescent probe bound to the target (HDAF-stained NG cells) obtained based on the fluorescence anisotropy ratio imaging data shown in FIGS. 22 to 25, respectively. 5 is a table showing the average value, the standard deviation, and the contrast value of the fluorescence anisotropy ratio in the region of FIG. As can be seen from this chart, the contrast value is larger when the time-gated method is used than when the steady-state light excitation is used, and when the polarization response correction is performed than when the polarization response correction is not performed. The value is large.

As described above, according to the depolarization imaging apparatus according to the present invention, a two-dimensional image of depolarization of fluorescence generated from a sample labeled with a fluorescent probe can be obtained under a microscope. Moreover, even under a microscope such as the depolarization imaging apparatus according to the present invention, highly accurate depolarization measurement can be performed by performing polarization response correction.

[0073]

As described above in detail, according to the present invention, the linearly polarized pulse excitation light for exciting the sample is output from the pulse excitation light source unit, and the polarization direction is rotated by the polarization direction rotating means, and the predetermined direction is rotated. And the irradiation optical system irradiates the sample from a predetermined direction. The fluorescence generated by irradiating the sample with the pulsed excitation light is
The first and second imaging planes which are input from substantially the same direction as the predetermined direction, are branched into first and second linear polarization components (p-polarized light and s-polarized light) orthogonal to each other, and are respectively different from each other. The image is formed by the first and second imaging units whose imaging timing is controlled by the control unit. Then, the two-dimensional image of the anisotropy of the fluorescence generated from the sample is converted into a linearly polarized light component of each of the first and second polarization directions of the fluorescence captured by the first and second imaging units by the image calculation unit. Is determined based on the image of

With this configuration, a two-dimensional image of the depolarization of the fluorescence generated by irradiating the fluorescently labeled sample with the excitation light can be detected under a microscope, and the target to which the fluorescent probe is bound can be detected. The existing position can be specified with high accuracy. Therefore, the behavior of the target in the sample can be analyzed, which can contribute to elucidation of various functions in cells, for example.

In particular, the images of the linearly polarized light components of the first and second polarization directions of the fluorescence imaged when the sample is irradiated with the pulsed excitation light having the first polarization direction, and the second Polarization response correction is performed based on the images of the linearly polarized light components of the first and second polarization directions of the fluorescence imaged when the sample is irradiated with the pulsed excitation light having the polarization direction, and the anisotropy of the fluorescence is obtained. By obtaining a two-dimensional image of the properties, when the molecular axes of the fluorescent probes in the sample are not oriented and are totally randomly distributed, the polarization response can be corrected appropriately, and the anisotropy of fluorescence can be improved. A two-dimensional image can be obtained with high accuracy.

Also, the first and second fluorescences captured when the sample at the predetermined rotation angle position with the predetermined direction as the center axis is irradiated with the pulsed excitation light having the first polarization direction. When the image of the linear polarization component of each polarization direction and the sample at the rotation angle position different from the predetermined rotation angle position by 90 degrees with respect to the center axis are irradiated with the pulse excitation light having the second polarization direction. By obtaining a two-dimensional image of fluorescence anisotropy by performing polarization response correction on the basis of the captured images of the linearly polarized light components of the first and second polarization directions of the fluorescence, the fluorescence probe in the sample can be obtained. When the molecular axes are oriented and distributed, polarization response correction can be suitably performed, and a two-dimensional image of fluorescence anisotropy can be obtained with high accuracy.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of depolarization of a free fluorescent label and a fluorescent label bound to a target.

FIG. 2 is an explanatory diagram of a method of obtaining a G factor in a conventional depolarization measuring device.

FIG. 3 is an explanatory diagram of how to obtain a G factor in the depolarization imaging apparatus according to the present invention.

FIG. 4 is an explanatory diagram of how to determine the G factor when the molecular axes of the fluorescent labels in the sample are oriented and distributed in the depolarization imaging apparatus according to the present invention.

FIG. 5 is a configuration diagram of a depolarization imaging apparatus according to the present invention.

FIG. 6 is a graph showing an attenuation curve (thick line) when the intensity of fluorescence generated from a sample is measured without polarization, and a response function (thin line) of the device under a microscope.

FIG. 7 shows a p-polarized component (thick line) and an s-polarized component (thin line) of fluorescence generated when excited by p-polarized pulsed excitation light.
It is a graph which shows each attenuation curve.

FIG. 8 shows a decay curve of the fluorescence anisotropy ratio obtained by performing the polarization response correction using the factor G (thick line), and a decay curve of the fluorescence anisotropy ratio obtained without performing the polarization response correction (thin line). FIG.

FIG. 9 shows the decay curve (thick line) of the fluorescence anisotropy ratio obtained by performing the polarization response correction in the depolarization imaging apparatus according to the present invention and the polarization response correction obtained by performing the polarization response correction in the conventional depolarization measurement apparatus. 4 is a graph showing the obtained fluorescence anisotropy ratio decay curve (thin line).

FIG. 10 shows the p-polarized component (thick line) and s of the fluorescence generated when the HDAF solution was excited with the p-polarized pulsed excitation light.
It is a graph which shows the attenuation curve of each polarization component (thin line).

FIG. 11 shows a p-polarized component of fluorescence generated when exciting HDAF-stained NG cells with p-polarized pulsed excitation light (thick line).
It is a graph which shows the attenuation curve of each of a s-polarized light component (small line).

FIG. 12: HDAF solution (thin line) and HDAF stained N
It is a graph which shows the decay curve of the fluorescence anisotropy ratio obtained by performing polarization response correction by G factor about the fluorescence generated when G cells (thick line) were excited.

FIG. 13 is a graph showing the results of simulation calculation of the fluorescence anisotropy ratio r by the time gate method for the HDAF solution (thin line) and the HDAF-stained NG cells (thick line).

FIG. 14 is a graph showing the results obtained as the contrast value of the fluorescence anisotropy ratio r in the depolarization measurement between a free fluorescent probe and a fluorescent probe bound to a target.

FIG. 15 shows a fluorescent probe (HDA bound to a target)
5 is a photograph of a halftone image in which a non-polarized light image of fluorescence generated from F-stained NG cells is displayed on a display.

FIG. 16 shows a free fluorescent probe (HDAF solution) and a fluorescent probe bound to a target (HDAF stain N
5 is a photograph of a halftone image in which imaging of a fluorescence anisotropy ratio (gate ON time: 0 ns) is displayed on a display for fluorescence generated from each of the (G cells).

FIG. 17 shows a free fluorescent probe (HDAF solution) and a fluorescent probe bound to a target (HDAF stain N
5 is a photograph of a halftone image in which imaging of a fluorescence anisotropy ratio (gate ON time: 1 ns) is displayed on a display for fluorescence generated from each of the (G cells).

FIG. 18 shows a free fluorescent probe (HDAF solution) and a fluorescent probe bound to a target (HDAF stain N
5C is a photograph of a halftone image in which fluorescence anisotropy ratio imaging (gate ON time: 2 ns) is displayed on a display for fluorescence generated from each of the G cells.

FIG. 19 is a table showing the results of a simulation calculation of the separation ability (contrast value) in depolarization measurement when a free fluorescent probe and a fluorescent probe bound to a target coexist.

FIG. 20: HDAF stained NG immersed in HDAF staining solution
It is a photograph of the halftone image which displayed the phase contrast image of the cell on the display.

FIG. 21: HDAF stained NG immersed in HDAF stain solution
It is the photograph of the halftone image which displayed the unpolarized fluorescence image of the cell on the display.

FIG. 22 is a photograph of a halftone image displayed on a display after correcting the fluorescence anisotropy ratio of the fluorescence anisotropy ratio of the HDAF-stained NG cells immersed in the HDAF staining solution when excited by the steady light.

FIG. 23 is a photograph of a halftone image displayed on a display without correcting polarization anisotropy imaging of the fluorescence anisotropy ratio of HDAF-stained NG cells immersed in an HDAF staining solution when excited by steady light.

FIG. 24 is a photograph of a halftone image displayed on a display after correcting the polarization anisotropy of the fluorescence anisotropy ratio of the HDAF-stained NG cells immersed in the HDAF staining solution in the case of the time gate method.

FIG. 25 is a photograph of a halftone image displayed on a display without correcting the polarization response of the imaging of the fluorescence anisotropy ratio of the HDAF-stained NG cells immersed in the HDAF staining solution in the case of the time gate method.

FIG. 26 shows a free fluorescent probe (HDAF solution) and a fluorescent probe bound to a target (HDAF stain N
(G cell) is a table showing the average standard deviation and the contrast value of the fluorescence anisotropy ratio in each region.

FIG. 27 is an explanatory diagram of auto-fluorescence removal in a time-resolved fluorescence microscope.

[Explanation of symbols]

1: pulse excitation light source, 2: polarizer, 3: half-wave plate,
4 ... Excitation light introduction optical system, 5 ... Dichroic mirror, 6
... Objective lens, 7 ... Sample, 8 ... Sample stage, 9 ... Band pass filter, 10 ... Polarizing beam splitter, 11,
12: imaging lens, 13, 14: image intensifier with gate function, 15: gate controller, 16
... Image operation unit.

Claims (3)

[Claims] A pulse excitation light source for outputting a linearly polarized pulse excitation light for exciting a sample; a polarization direction rotating means for rotating a polarization direction of the pulse excitation light output from the pulse excitation light source; An irradiation optical system for irradiating the sample with the pulse excitation light output from the polarization direction rotating unit from a predetermined direction; and inputting the fluorescence generated by irradiating the sample with the pulse excitation light from a direction substantially the same as the predetermined direction. A light receiving optical system that splits into linear polarization components of first and second polarization directions orthogonal to each other and forms images on first and second image planes different from each other, and the first image plane A first imaging unit that captures a linear polarization component of the first polarization direction of the fluorescence imaged on the second imaging direction; and a second polarization direction of the fluorescence imaged on the second imaging surface. Captures linear polarization component A second imaging unit that controls imaging timing of each of the first and second imaging units; and a first unit of the fluorescence imaged by each of the first and second imaging units. And an image calculation means for obtaining a two-dimensional image of the depolarization of the fluorescence generated from the sample based on the image of the linearly polarized light component of each of the second polarization directions. . 2. The method according to claim 1, wherein the calculating unit is configured to calculate the first and second polarization directions of the fluorescence imaged when the sample is irradiated with the pulsed excitation light having the first polarization direction. An image of a linearly polarized light component, and the first and second fluorescence images captured when the sample is irradiated with the pulsed excitation light having the second polarization direction.
2. The depolarization imaging apparatus according to claim 1, wherein a polarization response correction is performed based on the image of the linearly polarized light component of each polarization direction to obtain a two-dimensional image of the depolarization of the fluorescence. 3. The imaging means is imaged when the pulse excitation light having the first polarization direction is irradiated on the sample at a predetermined rotation angle position about the predetermined direction as a central axis. The image of the linearly polarized light component of each of the first and second polarization directions of the fluorescence and the sample at a rotation angle position different from the predetermined rotation angle position by 90 degrees with respect to the central axis. And performing polarization response correction based on images of the linearly polarized light components of the first and second polarization directions of the fluorescence imaged when the pulsed excitation light having the two polarization directions is irradiated. The depolarization imaging apparatus according to claim 1, wherein a two-dimensional image of the depolarization of the fluorescence is obtained.