JP6420910B2 - Microscope and image acquisition method - Google Patents

Description

  The present invention relates to a microscope and an image acquisition method.

A microscopic technique having a high spatial resolution exceeding the diffraction limit is known (for example, see Patent Document 1).
In Patent Document 1, a sample is irradiated with laser light having such intensity that fluorescence is saturated, and an image is generated by detecting only a signal corresponding to a saturation component of fluorescence using a modulation / demodulation technique. In the region where the fluorescence is saturated, the non-linearity of the fluorescence generation efficiency with respect to the excitation light is high, and it is possible to realize high spatial resolution by utilizing the fact that the spot diameter can be reduced by detecting this.

  More specifically, Patent Document 1 modulates the intensity of excitation light and demodulates it at a higher-order frequency. In the state where the fluorescence is not saturated, the time change of the fluorescence becomes equal to the time change of the excitation light. However, in the portion where the fluorescence is saturated, the time change of the fluorescence and the excitation light shows different aspects. Since the change appears as a higher-order frequency component of the modulated frequency as a result, only this higher-order frequency component is detected by demodulating with a lock-in amplifier.

  In Patent Document 1, only nonlinear components are obtained by irradiating excitation light having two or more intensities including the intensity at which fluorescence is saturated, acquiring images, and fitting the relationship of the fluorescence intensity to the excitation light intensity. An extraction method is also disclosed.

Japanese Patent No. 4487078 Japanese Patent No. 531595

Danielli et al. , Journal of Biomedical Optics 19 (8), 086006 (August 2014).

ここで、αはレーザ光に対する１次（１乗に比例）の成分の係数であり、βはレーザ光に対する２次（２乗に比例）の成分の係数である。なお、レーザ光の強度によっては３次（３乗に比例）以上の成分も発生するが、ここでは簡単のために２次までの成分で示している。蛍光の非飽和成分はαに寄与し、蛍光の飽和成分はβに寄与する。即ち、蛍光の飽和成分を検出するということは、βの成分を検出することに等しい。特許文献１に記載の方法では、ロックインアンプを利用して２ｆの成分のみを復調することにより、（２）式の最終項のみ、即ちβの成分のみをαの成分から分離して検出することができる。しかし、βは他の項にも含まれており、最終項に含まれる強度は全体の１／８である。即ち、周波数２ｆで復調して得られる蛍光飽和の信号は、実際に発生しているものの１／８であり、残り７／８は出力画像に寄与しない。そのため、特許文献１の方法では飽和信号の全てを取得することはできず、Ｓ／Ｎ比が悪いという課題がある。In the saturation component detection technique using modulation / demodulation described in Patent Document 1, a signal of a saturation component is only partially detected. For example, if the laser light is modulated at the frequency f, the intensity I _Ex (t) of the laser light and the intensity I _Fl (t) of the fluorescence can be expressed by the following expressions (1) and (2). .

Here, α is a coefficient of a first-order component (proportional to the first power) with respect to the laser light, and β is a coefficient of a second-order (proportional to the second power) component with respect to the laser light. Depending on the intensity of the laser beam, a component of the third order (proportional to the third power) or more may be generated. The fluorescent unsaturated component contributes to α, and the fluorescent saturated component contributes to β. That is, detecting the fluorescence saturation component is equivalent to detecting the β component. In the method described in Patent Document 1, only the last term of equation (2), that is, only the β component is detected separately from the α component by demodulating only the 2f component using a lock-in amplifier. be able to. However, β is also included in other terms, and the intensity included in the final term is 1/8 of the whole. That is, the fluorescence saturation signal obtained by demodulation at the frequency 2f is 1/8 of what is actually generated, and the remaining 7/8 does not contribute to the output image. For this reason, the method of Patent Document 1 cannot acquire all of the saturation signals, and there is a problem that the S / N ratio is poor.

  In addition, in the detection technique using the fitting described in Patent Document 1, it is necessary to perform a fitting process on each pixel, which requires an enormous amount of calculation and errors due to the fitting affect the image.

  The present invention has been made in view of the above-described circumstances, and provides a microscope and an image acquisition method capable of improving the S / N ratio and realizing higher spatial resolution by simple processing. It is aimed.

  In the first aspect of the present invention, signal light is generated by irradiation, and at least one of the irradiation lights has an irradiation intensity that saturates or nonlinearly increases or decreases the signal light, and two or more irradiation lights having different irradiation intensities are applied to the same portion of the sample. An irradiating unit that irradiates the sample separately, a signal light detecting unit that detects the signal light generated by irradiating the sample with the irradiation light, and a plurality of optical signals acquired by the signal light detecting unit. Based on the irradiation intensity and irradiation time of each corresponding irradiation light, the difference between a plurality of normalized optical signals obtained by normalization so that the light intensity components without saturation or non-linear increase / decrease coincide is calculated and obtained. It is a microscope provided with the image generation part which produces | generates a signal light image using the obtained difference signal.

  According to this aspect, the signal light detection unit acquires optical signals having intensities corresponding to the irradiation intensities of the respective irradiation lights separately irradiated to the same portion of the sample by the irradiation unit. In this case, since at least one irradiation light has an irradiation intensity that saturates or nonlinearly increases / decreases the signal light, a plurality of normalized light intensity components of these optical signals are matched by the image generation unit. By calculating the difference between the normalized optical signals, it is possible to efficiently extract only the saturation or nonlinear increase / decrease component of the signal light and generate a signal light image. Therefore, the S / N ratio can be improved and higher spatial resolution can be realized by a simple process that simply calculates the difference between a plurality of normalized optical signals. In this aspect, the signal light may be fluorescence.

In the above aspect, the irradiation unit sets the irradiation intensity and the irradiation time of each irradiation light so that the integrated values within the irradiation time of the irradiation intensity of each corresponding irradiation light are equal, so that the light It is good also as normalizing a signal.
With this configuration, the difference calculation process can be performed using the optical signal acquired by the signal light detection unit as it is as the normalized optical signal, and the difference calculation process can be simplified. Further, it is possible to avoid the narrowing of the dynamic range of the optical signal.

In the above aspect, the image generation unit may normalize the optical signal based on a ratio of integrated values within the irradiation time of the irradiation intensity of each corresponding irradiation light.
By comprising in this way, the saturation component of signal light can be extracted correctly, without adjusting the setting conditions of irradiation intensity and irradiation time of irradiation light.

In the above aspect, the irradiation unit irradiates the irradiation light that generates the signal light by a two-photon excitation method, and an integral value within the irradiation time of the square of the irradiation intensity of each corresponding irradiation light is The optical signal may be normalized by setting the irradiation intensity and irradiation time of each irradiation light so as to be equal.
With this configuration, the S / N ratio can be improved and higher spatial resolution can be realized by two-photon excitation observation with simple difference calculation processing.

In the said aspect, the said irradiation part irradiates the said irradiation light which generates the said signal light by a two-photon excitation method, and the said image generation part is the said irradiation time of the square of the irradiation intensity | strength of each corresponding said irradiation light. The optical signal may be normalized by the ratio of the integral values.
By configuring in this way, the S / N ratio is improved and higher spatial resolution is realized by two-photon excitation observation without taking time to adjust the setting conditions of irradiation intensity and irradiation time of irradiation light. Can do.

In the above aspect, the image generation unit calculates the difference for each line irradiated with the irradiation light, for each signal light image, for each pixel of the signal light image, or for each stack of the plurality of signal light images. It is good also as calculating.
By configuring in this way, when the difference is calculated for each signal light image or for each stack, the calculation process can be performed after forming an image for each irradiation light to some extent, and the difference process can be simplified. it can. In addition, when calculating the difference for each line or pixel, even if fluctuations in the irradiation time of the irradiation light, sample displacement, etc. occur due to the influence of changes in the environmental temperature, it is possible to make errors less likely to occur. .

In the above aspect, the image generation unit may include a lock-in amplifier that performs the difference calculation process, and the lock-in amplifier may calculate the difference for each pixel of the signal light image.
With this configuration, the difference calculation process can be performed easily and accurately using a commercially available lock-in amplifier.

In the above aspect, the irradiation unit separately irradiates the sample with three or more irradiation lights having different irradiation intensities, and the image generation unit performs a difference with respect to the plurality of normalized optical signals a plurality of times. It is good as well.
With this configuration, it is possible to generate a signal light image based on higher-order saturated components in the optical signal and realize higher spatial resolution.

In the above aspect, the image generation unit may have a deconvolution function.
With this configuration, even if a negative intensity component occurs during the difference calculation process, a signal light image that does not include the negative intensity component can be generated. Also, higher resolution can be realized by emphasizing the high-frequency component of the spatial frequency.

  In the second aspect of the present invention, signal light is generated by irradiation, and at least one of the irradiation lights has an irradiation intensity that saturates or nonlinearly increases / decreases the signal light, and two or more irradiation lights having different irradiation intensities are applied to the same portion of the sample. Irradiating separately, a signal light detecting step for detecting the signal light generated by irradiating the sample with each irradiation light, and a plurality of optical signals obtained by the signal light detecting step. A calculation step of calculating a difference between a plurality of normalized optical signals obtained by normalization so that the light intensity components that are not saturated or non-linearly increased or decreased are matched based on the corresponding irradiation intensity and irradiation time of each irradiation light And an image generation step of generating a signal light image using the difference signal obtained by the calculation step.

  According to this aspect, an optical signal having an intensity corresponding to the irradiation intensity of each irradiation light separately irradiated to the same portion of the sample by the irradiation step is acquired by the signal light detection step. In this case, since at least one irradiation light has an irradiation intensity that saturates or nonlinearly increases / decreases the signal light, a plurality of normalizations normalized so that the light intensity components of these optical signals coincide with each other in the calculation step Only the saturation or nonlinear increase / decrease component of the signal light can be efficiently extracted by the arithmetic processing of the difference of the optical signal. Therefore, a signal light image having a higher spatial resolution with an improved S / N ratio can be generated by the image generation step. In this aspect, the signal light may be fluorescence.

In the above aspect, the irradiation step sets the irradiation intensity and irradiation time of each irradiation light so that the integrated values within the irradiation time of the corresponding irradiation intensity of each irradiation light are equal. It is good also as normalizing a signal.
With this configuration, the difference calculation process can be performed by using the optical signal acquired in the signal light detection step as it is as the normalized optical signal, and the difference calculation process can be simplified. Further, it is possible to avoid the narrowing of the dynamic range of the optical signal.

In the above aspect, the image generation step may normalize the optical signal based on a ratio of integral values within the irradiation time of the corresponding irradiation intensity of the irradiation light.
By comprising in this way, the saturation component of signal light can be extracted correctly, without adjusting the setting conditions of irradiation intensity and irradiation time of irradiation light.

In the above aspect, the irradiation step irradiates the irradiation light that generates the signal light by a two-photon excitation method, and an integral value within the irradiation time of the square of the irradiation intensity of each corresponding irradiation light is The optical signal may be normalized by setting the irradiation intensity and irradiation time of each irradiation light so as to be equal.
With this configuration, it is possible to generate a signal light image having a higher spatial resolution with an improved S / N ratio by two-photon excitation observation by simple difference calculation processing.

In the above aspect, the irradiation step irradiates the irradiation light that generates the signal light by a two-photon excitation method, and the image generation step includes the irradiation time of the square of the irradiation intensity of each corresponding irradiation light. The optical signal may be normalized by the ratio of the integral values.
By configuring in this way, the S / N ratio is improved and higher spatial resolution is realized by two-photon excitation observation without taking time to adjust the setting conditions of irradiation intensity and irradiation time of irradiation light. Can do.

  According to the present invention, it is possible to improve the S / N ratio and realize higher spatial resolution with a simple process.

It is a typical whole block diagram which shows the fluorescence microscope which concerns on 1st Embodiment of this invention. It is a flowchart explaining the image acquisition method which concerns on 1st Embodiment of this invention. It is a figure which shows the relationship between the irradiation intensity | strength of a 1st laser beam, and a pulse width. It is a figure which shows the relationship between the irradiation intensity of a 2nd laser beam, and a pulse width. As a comparative example of the present embodiment, the first comparative fluorescence and the second comparative laser when the sample is irradiated with the first comparative laser light and the second comparative laser light that are changed only in the irradiation intensity at the same irradiation time. It is a figure which shows the PSF of fluorescence. The first fluorescence and the second fluorescence when the sample is irradiated with the first laser beam and the second laser beam whose irradiation intensity and pulse width are set so that the integrated values of the irradiation intensities of the respective laser beams within the irradiation time are equal. It is a figure which shows PSF of. It is a figure which shows the state from which only the nonlinear component (saturation component) which is the center part of PSF was extracted. It is a typical whole block diagram which shows the fluorescence microscope which concerns on 2nd Embodiment of this invention. As a comparative example of this embodiment, it is a figure which shows an example of the result of having carried out the two-photon excitation observation of the fluorescent bead of diameter 100nm like the conventional with the excitation light of wavelength 800nm. It is a figure which shows an example of the result of having carried out the two-photon excitation observation of the same bead as FIG. 6A in the same condensing position with the fluorescence microscope which concerns on this embodiment. It is the figure which showed the line profile of the bead shown with the number of 1 to 5 in FIG. 6A. It is a figure which shows the relationship between the irradiation intensity of a 3rd laser beam, and a pulse width. It is a typical whole block diagram which shows the fluorescence microscope which concerns on 4th Embodiment of this invention. It is a figure which shows the relationship between the irradiation intensity of a 1st laser beam and a 2nd laser beam, and a pulse width. It is a figure which shows the change of the fluorescence signal of each fluorescence generate | occur | produced by irradiation of a 1st laser beam and a 2nd laser beam. It is a typical whole block diagram which shows the fluorescence microscope which concerns on 5th Embodiment of this invention.

[First Embodiment]
A fluorescence microscope (microscope) and an image acquisition method according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the fluorescence microscope 1 according to the present embodiment has a configuration capable of observing the sample S by one-photon excitation. That is, the fluorescence microscope 1 includes a light source device (irradiation unit) 3 that emits laser light (irradiation light), an illumination optical system (irradiation unit) 5 that irradiates the sample S with laser light emitted from the light source device 3, and Based on a detection optical system (signal light detection unit) 7 that detects fluorescence (signal light) generated by irradiating the sample S with laser light, and a fluorescence signal (light signal) acquired by the detection optical system 7. A processing device (image generation unit) 9 such as a PC (Personal Computer) that generates a fluorescent image (signal light image) is provided.

The light source device 3 includes a laser light source 11 that generates laser light, and a light modulation unit 13 that modulates the laser light emitted from the laser light source 11.
The light modulation unit 13 is, for example, an acousto-optic element, and can modulate the laser light from the laser light source 11 into a pulse having a rectangular shape with a predetermined intensity and a predetermined pulse width.

  In the present embodiment, the light modulation unit 13 selectively emits the laser light from the laser light source 11 by switching to two laser lights having different irradiation intensities and pulse widths. Further, the light modulation unit 13 sets the irradiation intensity and pulse width (irradiation time) of these laser beams so that the integrated values within the irradiation time of the irradiation intensity of each laser beam in the exposure for one pixel are equal. It has become.

  The illumination optical system 5 includes a two-axis galvano scanner (hereinafter simply referred to as a scanner) 15 that two-dimensionally scans the laser light emitted from the light source device 3 and a pupil that condenses the laser light scanned by the scanner 15. While the projection lens 17, the imaging lens 19 that makes the laser light condensed by the pupil projection lens 17 substantially parallel light, and the laser light that has been made substantially parallel light by the imaging lens 19 irradiate the sample S, the sample S And an objective lens 21 that condenses the fluorescence generated when the S fluorescent material is excited.

  The scanner 15 includes a pair of galvanometer mirrors 16A and 16B that can swing around swinging axes that intersect each other. The scanner 15 is arranged so that the intermediate position between the pair of galvanometer mirrors 16A and 16B is conjugate to the pupil position of the objective lens 21. Further, the scanner 15 can scan the laser light in the X direction and the Y direction orthogonal to each other in accordance with the swing angle of each galvanometer mirror 16A, 16B.

  The detection optical system 7 includes a dichroic mirror 23 that branches the fluorescence that is collected by the objective lens 21 and returns to the optical path of the laser light from the optical path of the laser light, and a condensing lens 25 that collects the fluorescence branched by the dichroic mirror 23. And a pinhole 27 that restricts the passage of the fluorescence condensed by the condenser lens 25 and a detector 29 that detects the fluorescence that has passed through the pinhole 27.

  The dichroic mirror 23 transmits the laser light from the laser light source 11 that has passed through the light modulation unit 13 toward the scanner 15, while the laser light from the sample S passes through the objective lens 21, the imaging lens 19, and the pupil projection lens 17. The fluorescent light returning from the optical path is reflected toward the condenser lens 25.

The pinhole 27 is disposed at a position optically conjugate with the sample S, and constructs a confocal optical system. The pinhole 27 can pass only the fluorescence generated at the focal position of the objective lens 21 in the sample S.
The detector 29 is a photomultiplier tube (PMT), for example, and sends a fluorescence signal corresponding to the detected fluorescence intensity to the processing device 9.

  The processing device 9 scans the laser beam sent from the scanner 15 and the fluorescence signal sent from the detector 29 when fluorescence generated by irradiating the scanning beam with the laser beam is detected. Are stored in association with each other to generate a two-dimensional fluorescence image. The fluorescent image generated by the processing device 9 can be displayed on a display (not shown) or stored in a memory (not shown).

  In the present embodiment, the processing device 9 calculates the difference between the two fluorescent signals sent from the detector 29 corresponding to two laser beams having different irradiation intensities and pulse widths, and the obtained difference. A fluorescent image is generated using the signal. The light modulation unit 13 sets the irradiation intensity and the pulse width of the two laser beams so that the integrated values of the irradiation intensities of the respective laser beams within the irradiation time are equal to each other. The two fluorescence signals are normalized so that non-saturated components (light intensity components) in which saturation does not occur coincide with each other. Therefore, the two fluorescence signals output from the detector 29 become normalized fluorescence signals (normalized light signals), respectively.

Next, an image acquisition method according to the present embodiment will be described.
In the image acquisition method according to the present embodiment, as shown in the flowchart of FIG. 2, two laser beams, one of which has an irradiation intensity that saturates fluorescence and has mutually different irradiation intensity and pulse width, are the same on the sample S. It was acquired by the irradiation step S1 for irradiating the spot separately, the fluorescence detection step (signal light detection step) S2 for detecting the fluorescence generated by each laser beam being irradiated to the sample S, and the fluorescence detection step S2. An operation that calculates the difference between multiple normalized fluorescence signals obtained by normalizing multiple fluorescence signals based on the irradiation intensity and pulse width of each corresponding laser beam so that non-saturated non-saturated components match. Step S3 and image generation step S4 for generating a fluorescent image using the difference signal obtained in calculation step S3 are included.

The operation of the thus configured fluorescence microscope 1 and the image acquisition method will be described.
In order to observe the sample S with the fluorescence microscope 1 and the image acquisition method according to the present embodiment, the laser light is modulated by the light modulation unit 13, and the fluorescence images are respectively emitted by two laser lights having different irradiation intensity and pulse width. To get.

Specifically, first, as shown in FIG. 3A, the light modulation unit 13 has a pulse width of Δt1 at an intensity (I _EX1 ) at which the laser light generated from the laser light source 11 is not saturated for each pixel. To modulate. Hereinafter, the laser beam thus modulated is referred to as a first laser beam. In FIG. 3A, the vertical axis indicates the intensity of the laser beam, and the horizontal axis indicates the irradiation time. The same applies to FIGS. 3B, 7 and 9A.

  The first laser light emitted from the light modulation unit 13 passes through the dichroic mirror 23, is scanned by the scanner 15, and is collected by the pupil projection lens 17. Then, the first laser light is collimated by the imaging lens 19 and irradiated on the sample S by the objective lens 21 (irradiation step S1). Thereby, the first laser beam is scanned two-dimensionally on the sample S according to the swing angle of each galvanometer mirror 16A, 16B of the scanner 15.

  Fluorescence generated when the fluorescent material of the sample S is excited by scanning the first laser light is condensed by the objective lens 21 and is transmitted through the imaging lens 19, the pupil projection lens 17, and the scanner 15. Returning to the optical path, the dichroic mirror 23 branches off the optical path of the laser light.

  Then, the fluorescence is condensed by the condenser lens 25, and only the fluorescence generated at the focal position of the objective lens 21 in the sample S passes through the pinhole 27 and is detected by the detector 29 (fluorescence detection step S2). Thereby, in the processing apparatus 9, the fluorescence image of the sample S is produced | generated based on the scanning position of the laser beam corresponding to the fluorescence signal of the fluorescence acquired by the detector 29. FIG. The fluorescent image generated by the processing device 9 may be displayed on a display.

Next, as shown in FIG. 3B, the light modulation unit 13 has a pulse width of Δt2 shorter than Δt1 with an intensity (I _EX2 ) at which the laser light generated from the laser light source 11 is saturated for each pixel. To modulate. I _EX2 > I _EX1 . Hereinafter, the laser beam thus modulated is referred to as a second laser beam. The first laser light and the second laser light have an irradiation intensity (I _EX1 , I _EX2 ) and an irradiation time (Δt1, Δt2) such that the integrated values within the irradiation time of the irradiation intensity in the exposure for one pixel are equal to each other. ) Is set.

  The second laser beam is scanned two-dimensionally on the sample S as in the case of the first laser beam. Thereby, the fluorescence generated when the fluorescent substance of the sample S is excited is detected by the detector 29, and the fluorescent image of the sample S is generated by the processing device 9.

  Subsequently, the processing device 9 detects the fluorescence generated by the first laser beam and detects the fluorescence signal acquired by the detector 29 and the fluorescence generated by the second laser beam, thereby detecting the detector 29. The difference from the fluorescence signal acquired by the above is calculated (calculation step S3), and a fluorescence image is generated using the obtained difference signal (image generation step S4).

Here, the intensity of the fluorescence generated by the first laser light between the pixels (hereinafter referred to as the first fluorescence) is expressed by the following equation (3), and the fluorescence generated by the second laser light between the pixels. The intensity of (hereinafter referred to as second fluorescence) is represented by the following formula (4).

Since the first laser beam (I _EX1 ) is a condition that does not saturate the fluorescence, there is no β term in the equation (3). Further, since the time waveform of the laser is a rectangular wave, the time integral of the inside Delta] t ₁ and Delta] t ₂ is replaced with the product of the Delta] t ₁ and Delta] t _2, respectively. On the other hand, since the second laser light (I _EX2 ) has an intensity at which the fluorescence is saturated, the term β remains in the equation (4).

In this case, the light modulation section 13 causes the irradiation intensity and pulse of the two laser lights so that the integrated values within the irradiation time of the irradiation intensity of the first laser light and the second laser light in the exposure for one pixel become equal. By setting the width, the following equation (5) is established.

As a result, the processor 9 calculates the difference between the fluorescence signal of the first fluorescence and the fluorescence signal of the second fluorescence, thereby eliminating the linear term with α as shown in the following equation (6). Thus, only the non-linear component term of β, that is, only the saturated component of fluorescence can be extracted.

  In the equation (6), the β component of the equation (4) remains without being attenuated, which means that the present technique can detect the saturated component of fluorescence with high efficiency without any loss. Therefore, the processing device 9 generates a fluorescent image based on the difference signal obtained by the difference calculation process of the expression (6), so that a high resolution using only a saturated component of fluorescence has a high S / N ratio. Can be realized in the state.

Next, the contents described above using mathematical expressions will be conceptually described with reference to the drawings.
4A, 4B, and 4C show the fluorescence point image intensity distribution (PSF). 4A, 4B, and 4C, the vertical axis indicates the intensity of fluorescence, and the horizontal axis indicates the position. The same applies to FIGS. 6C and 9B. FIG. 4A shows, as a comparative example of the present embodiment, the first comparative fluorescence when the sample S is irradiated with the first comparative laser light and the second comparative laser light that are changed only in the irradiation intensity at the same irradiation time. And PSF of the second comparative fluorescence. The first comparison laser beam is modulated to an intensity at which the fluorescence is not saturated, and the second comparison laser beam is modulated to an intensity at which the fluorescence is saturated.

  The first comparison fluorescence and the second comparison fluorescence have different shapes depending on the presence or absence of saturation. However, since the irradiation intensity of the laser light is different, the fluorescence intensity is also greatly different. Extracting only the saturation component by the difference and improving the spatial resolution means that the tail portion of the PSF shown in FIG. 4A (that is, the fluorescence intensity portion where saturation is not generated) is efficiently brought close to zero by the difference. However, in the state of FIG. 4A, since the difference in intensity between the first comparative fluorescence and the second comparative fluorescence is large, the skirt does not become zero even if the difference calculation is performed, that is, a linear component remains.

  On the other hand, in this embodiment, as shown in FIG. 4B, the intensity of the first fluorescence and the second fluorescence is normalized by the light modulation unit 13 so that the non-saturated components (linear components) in which saturation does not occur coincide with each other. Therefore, the bottom portions of the PSFs shown in the figure match between the first fluorescence and the second fluorescence, and the central portion is in a state where the intensity is different due to saturation.

  Therefore, by calculating the difference between the fluorescence signal of the first fluorescence and the fluorescence signal of the second fluorescence by the processing device 9, the skirt (linear component) in FIG. 4B becomes zero, and as shown in FIG. 4C, the PSF Only the non-linear component (saturated component) which is the central part of can be efficiently extracted. Thereby, the width of the PSF can be narrowed to increase the spatial resolution.

  As described above, according to the fluorescence microscope 1 and the image acquisition method according to the present embodiment, the sample S is irradiated with two laser beams each having an irradiation intensity that saturates the fluorescence, and the processing device 9 causes the laser beams to be irradiated. By calculating the difference between the two fluorescence signals normalized so that the non-saturation components of the fluorescence signals corresponding to the same match, it is possible to efficiently extract only the fluorescence saturation component and generate a fluorescence image. Therefore, it is possible to improve the S / N ratio and realize a higher spatial resolution with a simple process that simply calculates the difference between two fluorescence signals.

  In the present embodiment, the irradiation intensity and pulse width of the first laser beam and the second laser beam are set in advance by the light modulator 13 so that the expression (5) is satisfied. Instead of this, the processing device 9 may normalize the fluorescence signals of the first fluorescence and the second fluorescence by multiplying the coefficients so that the expression (5) is substantially satisfied. Alternatively, both the irradiation intensity and pulse width setting of the first laser light and the second laser light, and the coefficient multiplication of the fluorescence signals of the first fluorescence and the second fluorescence may be performed. The fluorescence signal thus normalized becomes a normalized fluorescence signal.

In this case, for example, the ratio k of the integrated value within the irradiation time of the irradiation intensity of each laser beam is expressed by Equation (7).

  Then, as shown in equation (7), the processing device 9 sets a ratio k of the irradiation intensity of the laser beam to the integral value within the irradiation time, and multiplies the equation (3) of the first fluorescence by the ratio k. Therefore, the difference from the second fluorescence equation (4) may be calculated.

信号を係数倍するということは実質的に信号のダイナミックレンジを狭めることに繋がる反面、例えば非飽和状態の蛍光信号のＳ／Ｎ比が低い場合には、励起強度を高めてＳ／Ｎ比を向上させてから係数倍で正規化し、差分後のＳ／Ｎ比を高くすることもできる。By doing so, as shown in the following equation (8), it is possible to eliminate a linear term having α and extract only a nonlinear component term (saturated component) of β.

While multiplying the signal by a factor substantially reduces the dynamic range of the signal, for example, when the S / N ratio of a non-saturated fluorescent signal is low, the excitation intensity is increased to increase the S / N ratio. It is also possible to increase the S / N ratio after the difference by normalizing with a coefficient multiple after improvement.

In general, since β is a negative value, the direction of the difference calculation is subtracted from the fluorescence signal acquired in the saturated state from the fluorescence signal acquired in the non-saturated state, as shown in Equations (6) and (8). Although it is preferable to do so, it is not particularly limited.
Further, the pulse waveform of each laser beam does not have to be rectangular, and the equation (5) may be established at the time of difference.

  In the present embodiment, the first laser beam is set to an intensity at which the fluorescence is not saturated, but the first laser beam may also be set to an intensity at which the fluorescence is saturated. ) Can be deleted by the difference. In this case, the difference in terms of β is output.

  Further, in the present embodiment, up to the second-order term is shown as the nonlinear term, but the third-order and subsequent components may be generated. Even in this case, since the linear term is eliminated by the difference, all the second and higher order terms are detected.

  In addition, when actually acquiring a fluorescent image, the fluorescent image is first acquired with the first laser light having an intensity that does not saturate the fluorescence, and then the fluorescent image is acquired with the second laser light having an intensity that saturates the fluorescence. It is desirable to do. Since the irradiation intensity of the second laser light is stronger than that of the first laser light, the possibility of fluorescence fading during observation can be reduced by performing image acquisition with the first laser light first. . Note that since the irradiation time of the second laser light is short, the fading of fluorescence hardly occurs even in image acquisition using the second laser light.

In the present embodiment, the processing device 9 may have a deconvolution function and deconvolve the fluorescence image after the difference calculation.
For example, if the measurement condition error does not satisfy the equation (5), or if the coefficient M is set to be the following equation (9) instead of the equation (5), the first difference is calculated to satisfy this equation. When multiplying the fluorescence signal of the fluorescence or the fluorescence signal of the second fluorescence by the coefficient M or setting the irradiation intensity and pulse width of the laser beam by the light modulator 13, a negative intensity component is added to the PSF during the difference calculation. Will occur.

  If the PSF shape including the negative intensity component is grasped in advance by simulation or actual measurement, and the fluorescence image after the difference calculation is deconvoluted using the grasped PSF shape, the negative intensity component is not included. It becomes possible to restore the fluorescence image. In this case, the PSF including a negative component has a high spatial frequency, and the spatial resolution can be further improved by deconvolution.

  In the present embodiment, the difference is calculated for each fluorescent image, but instead, for example, the processing device 9 performs a stack of a plurality of fluorescent images, a line irradiated with laser light, Alternatively, the difference may be calculated at any timing for each pixel of the fluorescent image.

  When calculating the difference for each fluorescence image or for each stack of a plurality of fluorescence images, the calculation process can be performed after an image is formed to some extent for each excitation light, and the difference process can be simplified. is there. On the other hand, when the difference is calculated for each line of laser light or for each pixel of the fluorescent image, the irradiation time of the laser light may fluctuate due to the influence of changes in the environmental temperature or the stage on which the sample S is placed. Even if the sample S drifts and the position shift occurs, there is an advantage that an error can be hardly generated.

In the present embodiment, an acousto-optic element has been described as an example of the light modulation unit 13. However, any configuration may be used as long as the irradiation intensity and pulse width of laser light can be adjusted. For example, another element such as an electro-optical element may be used as the light modulation unit 13, or the laser light source 11 may be directly modulated without using the light modulation unit 13.
In the present embodiment, the biaxial galvano scanner is exemplified as the scanner 15. However, for example, the stage S may be held while holding the sample S by a stage, and the scanner 15 is limited to the biaxial galvano scanner. It is not a thing.

[Second Embodiment]
Next, a fluorescence microscope (microscope) and an image acquisition method according to the second embodiment of the present invention will be described below with reference to the drawings.
The fluorescence microscope 1 and the image acquisition method according to the present embodiment are different from the first embodiment in that two-photon excitation observation is performed instead of the one-photon excitation observation of the first embodiment.
In the description of the present embodiment, the same reference numerals are given to portions that share the same configuration as the fluorescence microscope 1 and the image acquisition method according to the first embodiment described above, and description thereof is omitted.

  As shown in FIG. 5, the fluorescence microscope 1 according to the present embodiment includes an ultrashort pulse laser such as a titanium sapphire laser as the laser light source 11. In addition, the fluorescent light emitted from the sample S and condensed by the objective lens 21 is not provided with the pinhole 27 and is branched from the optical path of the laser beam by the dichroic mirror 23 before entering the imaging lens 19. It is made to inject into.

  Also in the present embodiment, the light modulation unit 13 switches the laser light to two laser lights having different irradiation intensity and pulse width in a rectangular pulse, and within the irradiation time of the irradiation intensity of each laser light in exposure for one pixel. The irradiation intensity and pulse width of these two laser beams are set so that the integral values are equal.

In this case, the intensity of the first fluorescence generated by the first laser light during one pixel is represented by the following equation (10), and the intensity of the second fluorescence generated by the second laser light during one pixel is represented by the following ( 11)

Compared with equations (3) and (4) in the case of one-photon excitation, as shown in equations (10) and (11), in the case of two-photon excitation, the unsaturated component is irradiated with laser light. The difference is that it is proportional to the square of the intensity, and the saturation component is proportional to the fourth power of the irradiation intensity of the laser beam. By setting the irradiation intensity and pulse width of the two laser beams so that the integrated values within the irradiation time of the irradiation intensity of each laser beam in the exposure for one pixel are equalized by the light modulation unit 13, The following equation (12) is established as an equation corresponding to the equation (5).

Then, the difference between the fluorescence signal of the first fluorescence and the fluorescence signal of the second fluorescence is calculated by the processing device 9, so that the following equation (13) is obtained as in the above equation (6). , Α can be eliminated, and only the non-linear component term of β, that is, only the saturated component can be extracted.

  As described above, in the first embodiment, the time integration of the square of the irradiation intensity of the laser light between the pixels is made equal between the first laser light and the second laser light as in the condition of the expression (12). This is significantly different from the case of one-photon excitation.

  An example of the fluorescence image acquired by this embodiment, its comparative example, etc. are shown to FIG. 6A, 6B, 6C. FIG. 6A shows a result of two-photon excitation observation of a fluorescent bead having a diameter of 100 nm with a laser beam having a wavelength of 800 nm as in the past as a comparative example of the present embodiment. A plurality of beads are displayed in the fluorescence image.

  On the other hand, FIG. 6B shows the result of two-photon excitation observation of the same bead as in FIG. 6A at the same condensing position according to this embodiment. FIG. 6B shows that the size of the fluorescent bead is smaller than that of FIG. 6A, and the spatial resolution is improved.

  FIG. 6C shows a line profile of beads indicated by numbers 1 to 5 in FIG. 6A. FIG. 6C shows that the line profile is acquired at a position crossing the center of gravity of each bead, the position where the line profile is taken around the position of the center of gravity is rotated, each line profile is added and averaged, and this process is performed for each bead. The average of the line profile of 1 to 5 beads was taken. In FIG. 6C, the broken line is the line profile of the beads observed by the conventional two-photon observation method of FIG. 6A, and the solid line is the line profile of the beads observed by the two-photon excitation observation of this embodiment shown in FIG. 6B.

  As apparent from the line profile of FIG. 6C, according to the present embodiment, the spatial resolution can be improved as compared with the conventional two-photon excitation observation. Note that the line profile of FIG. 6C includes the size of the fluorescent beads, and is not the resolution of the microscope itself (PSF). That is, according to the fluorescence microscope 1 and the image acquisition method according to the present embodiment, the degree of improvement in PSF resolution is greater than the result of FIG. 6C.

  In the present embodiment, the fluorescence signal is normalized by the modulation of the laser beam by the light modulation unit 13, but instead, the processing device 9 emits the square of the irradiation intensity of each corresponding laser beam. It is good also as normalizing a fluorescence signal by the ratio of the integral value in time.

In this case, for example, the ratio k of the integral value within the irradiation time of the square of the irradiation intensity of each laser beam is expressed by equation (14).

  Then, as shown in the equation (14), the processing device 9 sets the integral value ratio k within the irradiation time of the square of the irradiation intensity of the laser light, and sets the ratio k in the equation (10) of the first fluorescence. What is necessary is just to calculate the difference with (11) Formula of 2nd fluorescence after multiplying.

In this way, only the nonlinear component term (saturated component) of β can be extracted as shown in the following equation (15).

[Third Embodiment]
Next, a fluorescence microscope (microscope) and an image acquisition method according to a third embodiment of the invention will be described below with reference to the drawings.
In the fluorescence microscope 1 and the image acquisition method according to this embodiment, the illumination optical system 5 separately irradiates the sample S with three or more laser beams having different irradiation intensities, and the processing device 9 has a plurality of normalized fluorescence. The difference from the first embodiment is that the difference with respect to the signal is performed a plurality of times. The configuration of the fluorescence microscope 1 is the same as that shown in FIG.
In the description of the present embodiment, the same reference numerals are given to portions that share the same configuration as the fluorescence microscope 1 and the image acquisition method according to the first embodiment described above, and description thereof is omitted.

In this embodiment, either one-photon excitation observation or two-photon excitation observation can be used, but one-photon excitation observation will be described.
The light modulation unit 13 selectively emits three laser beams having different irradiation intensities and pulse widths. For example, three laser beams are a first laser beam shown in FIG. 3A, a second laser beam shown in FIG. 3B, and a third laser beam shown in FIG. These are first fluorescence, second fluorescence, and third fluorescence, respectively. For example, the third laser light is assumed to have a pulse width of Δt3 shorter than Δt2 with an irradiation intensity stronger than that of the second laser light for each pixel.

  The light modulation unit 13 includes the first fluorescence including no saturation component, the second fluorescence including a saturation component exhibiting a second-order nonlinear response to the excitation laser, and the third fluorescence including a third-order saturation component. The irradiation intensity and pulse width of each laser beam are adjusted so that the integrated values within the irradiation time of the irradiation intensity of each laser beam in the exposure for one pixel are equal.

In this case, the intensity of the first fluorescence is expressed by the following expression (16), the intensity of the second fluorescence is expressed by the expression (17), and the intensity of the third fluorescence is expressed by the expression (18).

The pulse shapes of these three laser beams are rectangular, and the integration of the irradiation time is equal to multiplication by the pulse width.
The above equation (5) is established by setting the irradiation intensity and pulse width of each laser beam by the light modulator 13, and the difference between the fluorescence signal of the first fluorescence and the fluorescence signal of the second fluorescence is the same as in the first embodiment. The above equation (6) holds.

Similarly, the following equation (19) is established for the first fluorescence and the third laser light, and equation (20) is established by the difference between the fluorescence signal of the first fluorescence and the fluorescence signal of the third fluorescence.

In both equations (6) and (20), the linear term with α is eliminated.
In the processing device 9, the equation (20) is multiplied by I ² _EX2 Δt ₂ / I ² _EX3 Δt ₃ and then the difference calculation is performed with the components of the equations (6) and (20), thereby It becomes like this.

  In the equation (21), the β term whose fluorescence intensity is proportional to the square of the irradiation intensity of the laser beam is also deleted, and only the γ term proportional to the third power remains. This means that the second and subsequent nonlinear terms are detected in the first embodiment, whereas the third and subsequent nonlinear terms are detected in the third embodiment. Therefore, according to the present embodiment, further improvement in spatial resolution can be realized.

  This embodiment is not limited to the third order, and as long as the S / N ratio allows, the spatial resolution can be improved by increasing the order of nonlinear terms detected by increasing the number of times of image acquisition and difference. It is. Although it is assumed that the first fluorescence includes only a linear component, the second fluorescence includes up to a second-order nonlinear component, and the third laser light includes up to a third-order nonlinear component, both are higher-order nonlinear components. May be included. Further, the present embodiment is not limited to one-photon excitation, and can be used in two-photon excitation observation as in the second embodiment.

In the present embodiment, the processing device 9 may normalize the fluorescence signal based on the ratio of the integrated values within the irradiation time of the irradiation intensity of each corresponding laser beam.
In this case, for example, the ratio of the integrated values within the irradiation time of the irradiation intensity of the first laser beam and the second laser beam is set to the above equation (7), and the irradiation time of the irradiation intensity of the first laser beam and the third laser beam is used. The ratio of the integral values is the following equation (22).

  Then, the processing apparatus 9 multiplies the first fluorescence (16) equation by the ratio k of the laser light irradiation intensity shown in equation (7) to the integral value within the irradiation time, and then the second fluorescence equation (17). What is necessary is just to calculate the difference with these. By doing so, as shown in the above equation (8), it is possible to eliminate a linear term having α and extract only a nonlinear component term (saturated component) of β.

  Further, as shown in the equation (22), the processing device 9 sets the ratio l of the irradiation intensity of the laser beam to the integral value within the irradiation time, and multiplies the equation (16) of the first fluorescence by the ratio l. Therefore, the difference from the expression (18) of the third fluorescence may be calculated.

By doing so, as shown in the following equation (23), it is possible to eliminate a linear term with α and extract only a nonlinear component term (saturated component) including β and γ.

Then, the processing device 9 uses the ratio k and the ratio l with respect to the time integral value of the irradiation intensity of the laser beam as shown in the following expression (24) based on the expressions (8) and (23). Only high-order nonlinear components (saturated components) can be extracted.

[Fourth Embodiment]
Next, a fluorescence microscope (microscope) and an image acquisition method according to a fourth embodiment of the invention will be described below with reference to the drawings.
As shown in FIG. 8, in the fluorescence microscope 1 and the image acquisition method according to the fourth embodiment of the present invention, the processing device 9 includes a lock-in amplifier 31 that performs a difference calculation process. This is different from the first embodiment in that a difference is calculated for each pixel.
In the description of the present embodiment, the same reference numerals are given to portions that share the same configuration as the fluorescence microscope 1 and the image acquisition method according to the first embodiment described above, and description thereof is omitted.

  As shown in FIG. 9A, the light modulator 13 switches the first laser light and the second laser light a plurality of times within the time for detecting one pixel, and alternately irradiates the sample S at a specific frequency. Yes. In FIG. 9A, switching of each laser beam is repeated twice per pixel, but the number of times is not limited to this. Further, the light modulation unit 13 adjusts the irradiation intensity and pulse width of each laser beam so that the integrated values within the irradiation time of the irradiation intensity of each laser beam in the exposure for one pixel become equal. Yes.

  FIG. 9B shows changes in fluorescence signal of fluorescence generated by irradiation with each laser beam. The signal value of each pixel is an integrated value within each pixel time of the fluorescence signals of the first fluorescence and the second fluorescence generated by the first laser light and the second laser light. In FIG. The signal value of the second fluorescence is indicated by dots.

  Since the irradiation intensity of each laser beam and the pulse width are set by the light modulator 13, the expression (5) (equation (12) in the two-photon excitation) is satisfied, so the signal intensity of the first fluorescence and the second fluorescence The difference is only the nonlinear component. Further, the light modulation unit 13 alternately irradiates the sample S with the first laser beam and the second laser beam at a specific frequency as shown in FIG. 9A, thereby generating alternately at the specific frequency as shown in FIG. 9B. The first fluorescence and the second fluorescence to be detected are detected by the detector 29.

  The lock-in amplifier 31 is disposed between the detector 29 and the processing device 9 and detects the fluorescence signals of the first fluorescence and the second fluorescence shown in FIG. Demodulation is performed at a specific frequency of the modulation unit 13. Thereby, the difference between the fluorescence signal of the first fluorescence and the fluorescence signal of the second fluorescence can be extracted.

  According to the present embodiment, even if the fluorescence signal output from the detector 29 is arithmetically processed and the difference calculation is not performed by the processing device 9, the processing device 9 only processes the signal after passing through the commercially available lock-in amplifier 31. A fluorescence image using only a saturated component can be created. In this case, the processing device 9 only creates and displays a fluorescent image.

  Further, the saturation of the fluorescence has been described so far, but the present proposal can be used for other phenomena as long as the nonlinear response of the fluorescence signal due to the increase of the excitation light intensity. For example, in fluorescent proteins and synthetic dyes that cause fluorescence switching, isomerization occurs due to an increase in excitation light intensity. Since this is also a non-linear response (non-linear increase / decrease in fluorescence) to an increase in excitation light intensity, the spatial resolution can be improved by applying the present plan in the same manner as saturation. In this case, in the same manner as in each of the above embodiments in which each of the fluorescence signals is normalized so that the non-saturated components that are not saturated match each other, each of the fluorescence signals is matched so that the light intensity components that do not cause nonlinear increase / decrease match Should be normalized.

  Although the case where the signal light is fluorescence has been described so far, the present proposal is also useful in the case of other light detection in which the signal light nonlinearly responds by increasing the irradiation laser light intensity. It can also be used for reflection, absorption, scattering, and multiphoton effects. The effects on these phenomena are described below.

[Fifth Embodiment]
In this embodiment, the signal light is not the fluorescence emitted from the sample, but the reflected light, scattered light, transmitted light, or the light component generated by the multiphoton effect when the sample is irradiated with the laser light. First, the case where the signal light is reflected light or backscattered light will be described. The scattering described here refers to Mie scattering and Rayleigh scattering. An apparatus configuration diagram is shown in FIG.
In the description of the present embodiment, portions that share the same configuration as those of the fluorescence microscope 1 and the image acquisition method according to each of the embodiments described above are denoted by the same reference numerals and description thereof is omitted.

  FIG. 10 is substantially the same as FIG. 1 except that the dichroic mirror 23 is replaced with a beam splitter 33 and a dichroic filter 35 is inserted between the beam splitter 33 and the condenser lens 25. In FIG. 10, the light emitted from the laser light source 11 does not need to induce fluorescence of the sample S, and a component in which the laser light is reflected or backscattered by the sample S is detected as signal light. That is, the laser light incident on the sample S is reflected or backscattered by the sample S, and the reflected light or backscattered light is collected by the objective lens 21. Thereafter, the reflected light or backscattered light is deflected by the beam splitter 33, passes through the dichroic filter 35, passes through the pinhole 27, and is detected by the detector 29.

Here, when the intensity of the laser beam is increased, a nonlinear component of the laser beam is generated in the sample S. That is, when the wavelength of the laser beam is λ, a harmonic having a wavelength of λ / n (n: a natural number of 2 or more) is generated. The dichroic filter 35 is designed to transmit the same wavelength as that of the laser light and cut other wavelengths including nonlinear components generated in the sample S. Therefore, the signal that passes through the dichroic filter 35 and is detected by the detector 29 is only reflected light or backscattered light that does not contain a nonlinear component. Here, focusing on the intensity of the laser beam incident on the sample S, when the laser intensity is high and a nonlinear component is generated, the intensity of the component having the same wavelength as the laser beam is reduced by the amount of the nonlinear component. It will be. Therefore, in the state where the non-linear component is not generated, the intensity of the reflected light or the backscattered light from the sample S of the laser light is proportional to the intensity of the laser light incident on the sample S, but in the state where the non-linear component is generated. As a result, a non-linear decrease corresponding to the generation of the non-linear component occurs. This is the same effect as the above saturation of the fluorescence, and the equation is established even if the fluorescence intensity I _fl in the above equation is replaced with the reflection intensity I _Re and the scattering intensity I _Sc , respectively. That is, also in reflection and scattering, only a non-linear component can be extracted by acquiring a plurality of laser beams having different irradiation conditions and performing differential processing, and an image can be acquired based on the extracted nonlinear component. In this case, an image with improved spatial resolution can be acquired as in the case of fluorescence saturation.

  In the above description, reflected light or backscattered light is detected, but transmitted light or forward scattered light transmitted through the sample S may be detected. Even in this case, only the same wavelength component as the laser light is extracted by the dichroic filter 35 and detected by the detector 29. The same is true in that signal light is acquired by irradiating laser light with different irradiation conditions, and the spatial resolution is improved by extracting only nonlinear components by differential processing of the signal.

  The dichroic filter 35 may cut the same wavelength as the laser light and transmit other wavelengths. In that case, a component having a wavelength different from that of the laser light is detected by the detector 29. For example, hyper-Rayleigh scattering, harmonic generation, Raman scattering, coherent anti-Stokes Raman scattering (CARS), four-wave mixing, stimulated emission, difference frequency generation, sum generation, parametric fluorescence, or stimulated Raman scattering (SRS) Light may be detected. In this case, when the relationship of the laser beam intensity with respect to the signal beam intensity is linear as in Raman scattering, the integrated value within the irradiation time of the laser beam irradiation intensity is equal to the equation (5) in the first embodiment. Set the irradiation conditions. Further, when the relationship between the signal light intensity and the laser light intensity is non-linear as in the case of harmonic generation, the laser light irradiation intensity is raised to the power of the non-linear order as shown in the expression (12) of the second embodiment. Irradiation conditions are set so that the integral values of are equal. At this time, the nonlinear component extracted by the difference is a nonlinear component having a higher order than the order of the nonlinear effect for generating the signal light. That is, when considering SHG that is second-order harmonic generation, at least the order of the nonlinear effect for generating signal light is second-order, but the higher-order component extracted by the difference of the present plan is the third-order or later nonlinear component. It becomes. Further, the difference processing may be performed using the ratio k as in the equations (7) and (14), and only a higher-order nonlinear component is extracted by performing a plurality of differences as in the third embodiment. In addition, as in the fourth embodiment, it is possible to extract only the nonlinear component using a lock-in amplifier.

  In addition, a nonlinear increase component of the signal light generated by the nonlinear optical effect may be detected. For example, when the sample is a saturable absorber or the like, nonlinear signal light increases by increasing the laser light intensity. It is also possible to acquire only this non-linear increase component by the difference of the present plan. Moreover, it can utilize also for the detection of a photoacoustic signal like a nonpatent literature 1. In this case, as in the case of the present plan, the irradiation intensity and irradiation time of the laser beam irradiated on the sample are set under a plurality of conditions, and the difference between the acoustic signals detected by the transducer in each condition is taken, so that the laser light absorption is reduced. The spatial resolution can be improved by extracting high-order nonlinear components. As described above, the present scheme detects a nonlinear response component of a signal with respect to light irradiation, and the phenomenon may be anything and is not limited to the above. In addition, the light to irradiate the sample has been described as a laser beam so far. This is because a strong light source such as a laser is suitable for generating a non-linear response, and has an intensity that generates a non-linear response. Any light source may be used.

  As mentioned above, although each embodiment of this invention and its modification were explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design change of the range which does not deviate from the summary of this invention Etc. are also included.

1 Fluorescence microscope (microscope)
3 Light source device (irradiation part)
5 Illumination optical system (irradiation part)
7 Detection optical system (signal light detector)
9 Processing device (image generator)
S1 irradiation step S2 fluorescence detection step (signal light detection step)
S3 calculation step S4 image generation step

Claims (16)

An irradiation unit that generates signal light by irradiation, and at least one of the irradiation units has irradiation intensity that saturates or nonlinearly increases or decreases the signal light, and separately irradiates two or more irradiation lights having different irradiation intensities on the same portion of the sample; ,
A signal light detection unit for detecting the signal light generated by irradiating the sample with each of the irradiation lights, and
A plurality of optical signals acquired by the signal light detection unit are obtained by normalizing based on the irradiation intensity and irradiation time of each corresponding irradiation light so that the light intensity components that are not saturated or non-linearly increased or decreased coincide with each other. A microscope comprising: an image generation unit that calculates a difference between a plurality of normalized optical signals and generates a signal light image using the obtained difference signal.   The microscope according to claim 1, wherein the signal light is fluorescence.   The irradiation unit normalizes the optical signal by setting the irradiation intensity and the irradiation time of each irradiation light so that the integrated values within the irradiation time of the irradiation intensity of each corresponding irradiation light become equal. The microscope according to claim 1 or 2.   The microscope according to claim 1, wherein the image generation unit normalizes the optical signal based on a ratio of integral values within an irradiation time of irradiation intensity of each corresponding irradiation light.   The irradiation unit irradiates the irradiation light that generates the signal light by a two-photon excitation method, and the integral value within the irradiation time of the square of the irradiation intensity of each corresponding irradiation light becomes equal. The microscope according to claim 2, wherein the optical signal is normalized by setting an irradiation intensity and an irradiation time of each irradiation light. The irradiation unit irradiates the irradiation light to generate the signal light by a two-photon excitation method,
The microscope according to claim 2, wherein the image generation unit normalizes the optical signal based on a ratio of integral values within the irradiation time of a square of the irradiation intensity of the corresponding irradiation light.   The image generation unit calculates the difference for each line irradiated with the irradiation light, for each signal light image, for each pixel of the signal light image, or for each stack of the plurality of signal light images. The microscope according to claim 6.   The said image generation part is provided with the lock-in amplifier which performs the calculation process of the said difference, The said difference is calculated for every pixel of the said signal light image by this lock-in amplifier. microscope. The irradiation unit separately irradiates the sample with three or more irradiation lights having different irradiation intensities,
The microscope according to any one of claims 1 to 8, wherein the image generation unit performs a difference with respect to the plurality of normalized optical signals a plurality of times.   The microscope according to any one of claims 1 to 9, wherein the image generation unit has a deconvolution function. An irradiation step in which signal light is generated by irradiation, at least one of which has irradiation intensity that saturates or nonlinearly increases or decreases the signal light, and irradiates two or more irradiation lights having different irradiation intensities on the same part of the sample separately; ,
A signal light detection step for detecting the signal light generated by irradiating the sample with the irradiation light,
A plurality of signal light signals acquired by the signal light detection step are normalized based on the irradiation intensity and irradiation time of each corresponding irradiation light so that the light intensity components without saturation or non-linear increase / decrease coincide with each other. A calculation step for calculating a difference between a plurality of obtained normalized optical signals;
An image generation method including an image generation step of generating a signal light image using the difference signal obtained by the calculation step.   The image acquisition method according to claim 11, wherein the signal light is fluorescence.   The irradiation step normalizes the optical signal by setting the irradiation intensity and the irradiation time of each irradiation light so that the integrated values within the irradiation time of the irradiation intensity of each corresponding irradiation light become equal. The image acquisition method according to claim 11 or 12.   The image acquisition method according to claim 11, wherein the image generation step normalizes the optical signal based on a ratio of integral values within an irradiation time of irradiation intensity of each corresponding irradiation light.   The irradiation step irradiates the irradiation light that generates the signal light by a two-photon excitation method, and the integral value within the irradiation time of the square of the irradiation intensity of each corresponding irradiation light becomes equal. The image acquisition method according to claim 12, wherein the optical signal is normalized by setting an irradiation intensity and an irradiation time of each irradiation light. The irradiation step irradiates the irradiation light that generates the signal light by a two-photon excitation method,
The image acquisition method according to claim 12, wherein the image generation step normalizes the optical signal by a ratio of an integral value within the irradiation time of a square of the irradiation intensity of each corresponding irradiation light.

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