JP2010160264A - Laser microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a laser microscope for achieving accurate observation of two-photon excitation fluorescence while suppressing fluorescence by three-photon excitation. <P>SOLUTION: The laser microscope 1 includes: a laser beam source 2 emitting a pulse laser beam L; an objective lens 8 condensing the fluorescence generated on a sample A, while irradiating the sample A with the pulse laser beam L from the laser beam source 2; detectors 13a and 13b detecting the fluorescence condensed by the objective lens 8; and a dispersion compensating optical system 17 setting pulse width of the pulse laser beam L to be larger than minimum pulse width in which intensity of the fluorescence detected by the detectors 13a and 13b becomes maximum when adjustment of the pulse width of the pulse laser beam L is instructed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a laser microscope having a laser light source.

  Conventionally, a laser microscope that observes fluorescence generated in a specimen using pulsed laser light as excitation light is known (see, for example, Patent Documents 1 to 3). According to such a laser microscope, a high spatial resolution can be obtained particularly in the observation of a biological sample. In addition, by using a near-infrared wavelength pulse with high transmittance in a living tissue as excitation light, it is possible to realize minimally invasiveness to the sample and to observe the depth of the specimen.

Japanese Patent Publication No. 5-503149 JP 2006-106004 A JP 2007-127524 A

  However, according to the techniques disclosed in Patent Documents 1 to 3, when two-photon excitation observation is performed, fluorescence due to unintended three-photon excitation occurs and damages the biological specimen. Here, since the excitation light characteristic parameters related to the excitation efficiency of the two-photon excitation and the excitation efficiency of the three-photon excitation are common, when the excitation light is adjusted so as to reduce the three-photon excitation efficiency without darkness, the original observation is performed. There is an inconvenience that the observation accuracy of the two-photon excitation fluorescence to be reduced also decreases.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a laser microscope capable of accurately observing two-photon excitation fluorescence while suppressing fluorescence due to three-photon excitation.

In order to achieve the above object, the present invention employs the following means.
The present invention is directed to a laser light source that emits pulsed laser light, an object lens that irradiates the sample with pulsed laser light from the laser light source, and condenses the fluorescence generated in the sample, and is focused by the objective lens. Detection means for detecting fluorescence, adjustment instruction means for instructing adjustment of the pulse width of the pulse laser light, and when the adjustment instruction means instructs adjustment of the pulse width of the pulse laser light, the pulse laser light A laser microscope is used that includes a pulse width adjusting means for setting the pulse width of the above to be larger than the minimum pulse width at which the intensity of the fluorescence detected by the detecting means is maximized.

  According to the present invention, when adjustment of the pulse width of the pulse laser beam is instructed by the adjustment instruction unit, the pulse width of the pulse laser beam is larger than the minimum pulse width at which the fluorescence intensity is maximized by the pulse width adjustment unit. Since it is set, the intensity of the three-photon excitation fluorescence from the specimen can be reduced to reduce damage to the specimen. At this time, by setting the pulse width of the pulsed laser beam so as to ensure the intensity of the two-photon excitation fluorescence suitable for observation, the specimen by the two-photon excitation fluorescence is suppressed while suppressing damage to the specimen by the three-photon excitation fluorescence. Can be accurately observed.

  In the above invention, a branching unit for branching fluorescence condensed by the objective lens into two-photon excitation fluorescence and three-photon excitation fluorescence is provided, and the detection unit detects the two-photon excitation fluorescence branched by the branching unit. A two-photon fluorescence detection unit that detects the three-photon fluorescence that is branched by the branching unit.

  In this way, the two-photon excitation fluorescence and the three-photon excitation fluorescence generated in the sample can be detected separately and simultaneously, and the adjustment time of the pulse width of the pulsed laser beam is shortened to damage the sample. Can be reduced.

  In the above invention, the pulse width adjusting means may be configured so that the absolute value of the rate of change of the intensity of the three-photon excitation fluorescence detected by the three-photon fluorescence detector is equal to or less than a predetermined threshold value. It is good also as adjusting.

  The intensity of the three-photon excitation fluorescence is inversely proportional to the square of the pulse width of the pulsed laser light that is the excitation light. Therefore, by adjusting the absolute value of the rate of change of the intensity of the three-photon excitation fluorescence below a predetermined threshold by the pulse width adjusting means, the pulse width of the pulse laser beam is adjusted so that the intensity of the three-photon excitation fluorescence does not increase rapidly. It is possible to suppress damage to the specimen due to three-photon excitation fluorescence.

  In the above invention, the pulse width adjusting means may be configured such that the absolute value of the rate of change of the intensity of the two-photon excitation fluorescence detected by the two-photon fluorescence detection unit is equal to or greater than a predetermined threshold value. It is good also as adjusting.

  The intensity of two-photon excitation fluorescence is inversely proportional to the pulse width of the pulsed laser light that is excitation light. Therefore, by setting the absolute value of the rate of change of the intensity of the two-photon excitation fluorescence to a predetermined threshold or more by the pulse width adjusting means, the pulse width of the pulse laser beam is set so as to dramatically improve the intensity of the two-photon excitation fluorescence. It is possible to adjust, and it becomes possible to improve the observation accuracy by the two-photon excitation fluorescence.

  In the above invention, the pulse width adjusting means is configured such that the ratio of the intensity of the three-photon excitation fluorescence detected by the three-photon fluorescence detection unit to the intensity of the two-photon excitation fluorescence detected by the two-photon fluorescence detection unit is a predetermined threshold value. It is good also as adjusting the pulse width of the said pulse laser beam so that it may become the following.

  By doing so, the pulse width of the pulse laser beam can be adjusted by the pulse width adjusting means based on the balance between the intensity of the two-photon excitation fluorescence and the intensity of the three-photon excitation fluorescence, and two-photons suitable for observation. An increase in three-photon excitation fluorescence can be suppressed while ensuring the intensity of excitation fluorescence. As a result, it is possible to accurately observe the specimen by the two-photon excitation fluorescence while suppressing damage to the specimen by the three-photon excitation fluorescence.

  In the above-described invention, each image is obtained from the operation unit that operates the pulse width adjusting unit, the two-photon excitation fluorescence detected by the two-photon fluorescence detection unit, and the three-photon excitation fluorescence detected by the three-photon fluorescence detection unit. It is good also as providing the image generation part which produces | generates, and the display part which displays the image produced | generated by this image generation part.

  By doing so, it is possible to generate a two-photon excitation fluorescence image and a three-photon excitation fluorescence image by the image generation unit, and display these images on the display unit. By operating the width adjusting means, the pulse width of the pulse laser beam can be adjusted so that each image has a desired intensity.

  According to the present invention, there is an effect that two-photon excitation fluorescence can be accurately observed while suppressing fluorescence due to three-photon excitation.

1 is a schematic configuration diagram of a laser microscope according to a first embodiment of the present invention. It is a schematic block diagram of the dispersion correction optical system of FIG. 2A and 2B are diagrams for explaining the operation of the dispersion correction optical system of FIG. 1, in which FIG. 1A shows a case where the optical path length is shortened, and FIG. It is a figure explaining the effect | action of the laser microscope of FIG. It is a figure explaining the adjustment method of a pulse width, (a) is a case where 2 photons> 3 photons, (b) is a case where 2 photons <3 photons, (c) is a case where the intensity | strength of each fluorescence is normalized. is there. It is a schematic block diagram of the laser microscope which concerns on a 1st modification. It is a schematic block diagram of the dispersion | distribution adjustment system which concerns on a 2nd modification. It is a schematic block diagram of the laser microscope which concerns on a 4th modification. It is a schematic block diagram of the laser microscope which concerns on the 2nd Embodiment of this invention. It is a figure explaining the effect | action of the laser microscope of FIG.

[First Embodiment]
A laser microscope according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the laser microscope 1 of the present embodiment includes a laser light source 2 that emits pulsed laser light L, and a fluorescence microscope that irradiates the sample A with the pulsed laser light L and observes the fluorescence F that is obtained. A main body 3 and an introduction optical system 4 for introducing pulsed laser light L emitted from the laser light source 2 into the microscope main body 3 are provided.

  The fluorescence microscope main body 3 includes a galvanometer mirror 5, a pupil projection lens 6, an imaging lens 7, an objective lens 8, an excitation dichroic mirror (branching means) 9, a condenser lens 10, a barrier filter 11, a fluorescence A branch dichroic mirror 12 and detectors (detection means) 13a and 13b are provided. In the figure, reference numeral 14 denotes a mirror, reference numeral 15 denotes a band-pass filter, and reference numeral 16 denotes a condenser lens.

The galvanometer mirror 5 two-dimensionally scans the pulse laser beam L incident from the introduction optical system 4.
The pupil projection lens 6 focuses the pulsed laser light L scanned by the galvanometer mirror 5 to form an intermediate image.

The imaging lens 7 condenses the pulsed laser light L on which the intermediate image is formed by the pupil projection lens 6 and converts it into parallel light.
The objective lens 8 condenses the pulsed laser light L converted into parallel light by the imaging lens 7 to form an image on the sample A, and also emits the fluorescence F emitted when the fluorescent material is excited in the sample A. Condensed.

The excitation dichroic mirror 9 branches the fluorescence F condensed by the objective lens 8 from the pulsed laser light L.
The condensing lens 10 condenses the fluorescence F branched by the excitation dichroic mirror 9.
The barrier filter 11 removes the pulsed laser light L component in the fluorescence F collected by the condenser lens 10.

The fluorescence branching dichroic mirror 12 branches the fluorescence F that has passed through the barrier filter 11 for each wavelength.
The detectors 13a and 13b are, for example, photomultiplier tubes (PMT) and detect the fluorescence F branched by the wavelength by the fluorescence branching dichroic mirror 12, respectively.

  The introduction optical system 4 includes a dispersion compensation optical system (pulse width adjusting means) 17 that adjusts the pulse time width of the pulsed laser light L, an alignment adjusting device 18 that can change the optical axis and angle of the pulsed laser light L, A beam shaping optical system 20 for adjusting the beam diameter of the pulse laser L incident on the fluorescence microscope main body 3, an acoustooptic device 19 disposed between the alignment adjustment device 18 and the beam shaping optical system 20, and the pulse laser L And a microscope incident correction device 21 for adjusting the beam diameter of the microscope.

The alignment adjusting device 18 causes the pulse laser beam L, whose pulse time width is adjusted by the dispersion compensation optical system 17, to enter the mirror, and adjusts the optical axis and angle of the pulse laser beam L by adjusting the position and angle of the mirror. It can be changed.
The acoustooptic device 19 is, for example, an AOM (Acoustic Optical Modulator), and performs on / off or intensity modulation of the pulsed laser light L.

The beam shaping optical system 20 is a beam expander for narrowing the light beam of the pulsed laser light L so that it enters the effective range of the crystal of the acoustooptic device 19 without leaking.
The microscope incident correction device 21 is an adjustment mechanism for increasing the spatial density of photons by adjusting the diameter of the light beam incident on the objective lens and matching it with the pupil diameter of the objective lens 8. Further, the microscope incident correction device 21 emits the pulsed laser light L emitted from the acoustooptic device 19 so that it enters the pupil position of the objective lens 8 of the microscope body 3 with a light beam diameter equivalent to the pupil diameter. This is a beam expander that adjusts the beam shape and beam divergence of the pulsed laser beam L.

  Here, the probability that the multiphoton excitation effect such as the two-photon excitation effect and the three-photon excitation effect in the sample A can be obtained increases as the spatial and temporal density of the photons at a desired position to be excited increases. Therefore, as a means for increasing the temporal density of photons, a laser light source 2 that emits an extremely short pulse laser having a very high pulse time width of about 100 fs is used as an excitation light source.

  Further, in order to increase the spatial density of the photons, the excitation light incident on the sample A is condensed on a minute region by the objective lens 8. In order to increase the light density at the condensing point, it is necessary to condense at the smallest possible point. The pulsed laser light L can be condensed the smallest when the diameter of the light beam incident on the objective lens 8 is the same as the pupil diameter of the objective lens 8.

  On the other hand, unlike a general laser light source having a very narrow wavelength spectrum width, an ultrashort pulse laser beam used as excitation light has a somewhat wide wavelength spectrum. Here, the speed at which the light passes through the refractive index medium depends on the wavelength of the light, and the faster the longer the light, the slower the shorter the light. Therefore, when the pulsed laser light L passes through the optical element existing from the light source to the sample A, the short wavelength component included in the pulse is delayed in time compared to the long wavelength component. Hereinafter, this delay is referred to as group velocity dispersion.

  Due to the influence of the group velocity dispersion, light having a pulse width of about 100 fs at the light source spreads at the sample A to a pulse width of 200 fs or more. As the time width of the pulse increases, the temporal density of photons decreases, and the excitation efficiency of the multiphoton excitation effect decreases. Therefore, if the long wavelength component is adjusted in advance so as to be delayed with respect to the short wavelength component in advance, it is the group velocity dispersion when passing through the optical element. It becomes possible to irradiate the sample A with pulsed light whose wavelength components coincide in time and have a narrow pulse width.

  The dispersion compensation optical system 17 applies negative group velocity dispersion by passing the pulsed laser light L incident from the laser light source 2 and adjusts the pulse time width of the pulsed laser light L. As shown in FIG. 2, the dispersion correction optical system 17 is composed of two prisms 22 and 23 and a mirror 24, and the group velocity dispersion given to the pulse laser beam L by changing the interval between the two prisms. Can be adjusted.

  Specifically, the dispersion correction optical system 17 applies in advance the group velocity dispersion in the opposite direction to the pulse laser light L by the same amount as the group velocity dispersion generated in the optical system from the laser light source 2 to the sample A. Thereby, it is possible to prevent the pulse width of the pulse laser beam L from expanding in the sample A, and to increase the temporal density of photons.

  The dispersion correction optical system 17 operates by inputting changes in the optical system such as the sample A and the objective lens by an input device (adjustment instruction means) (not shown). Further, a change in the optical system such as the sample A or the objective lens may be detected by a sensor or the like, and the dispersion correction optical system 17 may be operated based on the detection result.

The operation of the dispersion correction optical system 17 configured as described above will be described with reference to FIGS. 3 (a) and 3 (b).
The pulsed light L incident on the prism 22 is dispersed for each wavelength and then incident on the prism 23. In the prism 23, the optical path length through which the long wavelength component (indicated by a chain line in the drawing) of the pulsed light L passes is longer than the optical path length through which the short wavelength component (indicated by the solid line in the drawing) passes. The pulsed light exiting the prism 23 is reflected by the mirror 24 and returns on the same optical path.

  Here, in a medium having a high refractive index, the speed of light is slower than in air, so that the optical path length through which the long wavelength passes when passing through the prism 23 is longer than the optical path length through which the short wavelength passes. Therefore, by operating the dispersion correction optical system 17, it becomes possible to generate pulsed laser light in which the long wavelength component is delayed in time.

  Here, as shown in FIG. 3A, when the distance between the two prisms is small, the optical path length through which the long wavelength component passes and the optical path length through which the short wavelength component passes when the pulsed light passes through the prism 23. There is little difference. On the other hand, as shown in FIG. 3B, when the distance between the two prisms is large, the difference between the optical path length through which the long wavelength component passes and the optical path length through which the short wavelength component passes when passing through the prism 23 is large. . In this way, by adjusting the prism interval, the dispersion correction amount applied in advance to the pulsed light L can be adjusted.

  The dispersion correction amount to be adjusted differs depending on the wavelength of the pulsed laser light L used as the excitation light and the configuration of the optical element from the laser light source 2 to the sample A. By adjusting the prism interval in this way, It is possible to increase the excitation efficiency of the multiphoton excitation effect by adjusting the pulse width so that it does not spread. When the prism interval is changed, the optical axis of the pulsed laser beam L also moves. Therefore, the prisms 22 and 23 and the mirror 24 are swung as indicated by the arrows shown in FIG. Need to adjust their angles.

  The pulse laser beam L whose dispersion amount has been corrected as described above enters the microscope body 3 and is two-dimensionally scanned by the galvanometer mirror 5 or positioned at a predetermined irradiation position. Then, the pulsed laser light L is condensed at a predetermined irradiation position of the sample A through the pupil projection lens 6, the imaging lens 7 and the objective lens 8. Thus, a multiphoton excitation effect occurs at the beam waist position of the pulsed laser light L focused on the sample A.

  The multi-photon excitation effect includes two-photon excitation fluorescence that emits fluorescence when excited by simultaneously absorbing two photons, three-photon excitation fluorescence that emits fluorescence when simultaneously absorbed by three photons, or Phenomena such as n-photon excitation fluorescence that emits fluorescence when excited by simultaneously absorbing n (n> 3) photons are included, and these are collectively referred to as multi-photon excitation fluorescence.

  After the multiphoton excitation fluorescence F generated in the sample A is collected by the objective lens 8, branched from the pulse laser beam L by the excitation dichroic mirror 9, and transmitted through the mirror 14, the collection lens 10 and the barrier filter 11. The light is branched for each wavelength by the fluorescence branching dichroic mirror 12 and detected by the detectors 13a and 13b.

  By the way, it is known that the fluorescence wavelength of the multiphoton excitation fluorescence is slightly longer than λ / n, where λ is the wavelength of the pulsed laser light L and n is the number of photons absorbed for excitation. Therefore, when the multiphoton excitation fluorescence generated from the sample A is mixed with two-photon excitation fluorescence and fluorescence excited by simultaneous absorption of three or more photons, the wavelengths of the respective fluorescences are different. .

  For example, when observing the two-photon excitation fluorescence when the wavelength of the pulse laser beam L is 720 nm and the fluorescence reagent applied to the sample A is excited by two-photon excitation, the two-photon excitation fluorescence depends on the type of the reagent. In many cases, the fluorescence wavelength is from 400 nm to 500 nm.

  On the other hand, a specific molecule itself in the cell tissue of sample A absorbs three photons of the pulsed laser light L at the same time to be in an excited state, and autofluorescence that generates three-photon excitation fluorescence at a fluorescence wavelength of 300 nm to 400 nm occurs. It is known that there are things. The occurrence of such three-photon excitation fluorescence is of interest as a research object, but also causes unintended changes and damage to the specimen. For the purpose of observing only two-photon excitation fluorescence from a sample, It is preferable not to generate three-photon excitation fluorescence as much as possible.

  In this case, the fluorescence branching dichroic mirror 12 uses an optical characteristic that transmits the wavelength of the two-photon excitation fluorescence while reflecting the wavelength of the three-photon excitation fluorescence. The photon excitation fluorescence and the three-photon excitation fluorescence are branched by the excitation branching dichroic mirror 12 and detected separately by the two detectors 13a and 13b, respectively, and the fluorescence intensity of the two-photon excitation fluorescence and the fluorescence intensity of the three-photon excitation fluorescence are independent. At the same time.

Alternatively, if the pulse laser L is scanned two-dimensionally by the scanner 5, a two-photon excitation fluorescence image and a three-photon excitation fluorescence image can be separately and simultaneously acquired.
In this way, while observing the two-photon excitation fluorescence from the specimen, it is also possible to measure how much three-photon excitation fluorescence that is not intended is generated.

Here, the excitation of two-photon excitation effect efficient I ₂ and 3 excitation efficiency I ₃ of excitation effect has been found to be respectively represented by the following equations.
I ₂ = Qσ ₂ CP _ave ² / F _p T _p (1)
I ₃ = Qσ ₃ CP _ave ³ / (F _p T _p ) ² (2)

In the above equation, Q is the quantum yield (%) of light emission depending on the specimen, σ ₂ is the absorption cross section of 2 photons (cm ⁴ s ² photon ^-1 molecule ^-1 ), and σ ₃ is the absorption cutoff of 3 photons. Area (cm ⁶ s ² photon ^-1 molecule ^-1 ), C is the number density of molecules depending on the sample (molecule ^-3 ), P _ave is the laser average power and laser average power, F _p is pulse repetition frequency which depends on the laser beam (Hz), T _p denotes a laser beam, the amount of dispersion of the device, the pulse width that depends on the dispersion correction amount (sec).

The excitation efficiency I ₂ of the two-photon excitation effect is proportional to the square of Average Power and inversely proportional to the pulse width, as shown in Equation (1). On the other hand, the excitation efficiency I ₃ of the three-photon excitation effect is proportional to the third power of the average power and inversely proportional to the square of the pulse width, as shown in the equation (2).
According to this, the excitation efficiency of the two-photon excitation effect and the three-photon excitation effect are both more efficient as the pulse width T _p of the excitation pulse light is smaller, and more efficient as the average intensity P _ave of the excitation pulse light is larger. C., also, it can be seen efficiency increases the repetition frequency F _p of the pumping pulse light is small is good.

Incidentally, in general, when observing the multiphoton excitation fluorescence, the pulse width T _p may be adjusted to optimize the conditions to be a minimum as possible in the device configuration, so the excitation efficiency is increased Often observed. As for the average pulse intensity, it is better if the excitation efficiency alone is considered. However, it is desirable that the pulse average intensity is weak in consideration of damage to the biological material. Therefore, the setting varies depending on the specimen and the type of observation.

The tendency of the change in excitation efficiency when the pulse width T _p , the average intensity P _ave , and the repetition frequency F _p are changed is different between the two-photon excitation effect and the three-photon excitation effect. Specifically, the amount of change in the two-photon excitation efficiency and the three-photon excitation efficiency when the pulse width T _p , the average intensity P _ave , and the repetition frequency F _p are changed by ΔT _p , ΔP _ave , and ΔF _p are as follows: become that way.

ΔT _p (2 photon variation: 3 photon variation) = (＝ 1 / ΔT _p : 1 / ΔT _p ² )
ΔP _ave (2 photon variation: 3 photon variation) = (∝ΔP _ave ² : ∝ΔP _ave ³ )
ΔF _p (2 photon variation: 3 photon variation) = (＝ 1 / ΔF _p : _ｐ1 / ΔF _p ² )

According to this, for example, when the pulse width is increased by ΔT _p from the minimum pulse width possible with the device configuration, the efficiency of the two-photon excitation effect is ΔT _p as shown in FIG. However, the efficiency of the three-photon excitation effect is attenuated in inverse proportion to ΔT _p ² , and the three-photon excitation effect is attenuated more than the two-photon excitation effect for the same amount of change. . The same applies to the average intensity and the repetition frequency, and the three-photon excitation effect changes more greatly for the same change than the two-photon excitation effect.

Here, a method of adjusting the pulse width in a conventional laser microscope, the pulse width T _p at the beam waist of the pulse laser light L condensed into a specimen A was to adjusted as small as possible. Therefore, although the excitation efficiency of the two-photon excitation effect is optimized, the excitation efficiency of the three-photon excitation effect is also optimized at the same time, and an unintended three-photon excitation effect is likely to occur.

On the other hand, according to the laser microscope 1 according to the present embodiment, the dispersion compensation optical system 17 is operated to change the direction in which the pulse width T _p is increased, or the average intensity P _ave of the pulsed light L is reduced. By doing so, the excitation efficiency of the three-photon excitation effect can be attenuated more than the excitation efficiency of the two-photon excitation effect. However, since these excitation efficiencies greatly change with a slight change in the pulse width T _p and the average intensity P _ave , in order to prevent the two-photon excitation effect of the core from being greatly attenuated. It is preferable that the adjustment of the pulse width T _p and the average intensity P _ave can be finely adjusted with high accuracy.

In the method of reducing the average intensity P _ave of the pulsed laser light L to suppress the three-photon excitation, the absolute number of photons of the pulsed laser L incident on the sample is reduced, and the sample becomes deep due to the opacity of the sample. The number of photons that reach the surface also decreases as it goes from the sample surface to the deep portion, and as a result, the observation accuracy at the deep portion of the sample, which is a merit of the two-photon excitation observation, also decreases.

On the other hand, in the method of widening the pulse width T _p, the absolute number of photons of the pulse laser L incident on the specimen without changing, so are changed only temporal photon density, two-photon excitable deep reach Sex is not lost. Therefore, in order to suppress the three-photon excitation effect, it is preferable to perform adjustment to increase the pulse width T _p rather than adjustment to reduce the average intensity P _ave .

Therefore, in the laser microscope 1 according to the present embodiment, a method is adopted in which the pulse width T _p is adjusted by the dispersion correction optical system 17 to suppress the generation of the three-photon excitation effect. That is, by adjusting the distance interval between the prisms 22 and 23 optimally adjusted for each wavelength of the pulsed laser light L so that the interval is intentionally changed from the optimum state, the pulse width T _p is continuously set. Can be expanded little by little.

Specifically, the dispersion correction optical system 17, when the adjustment of the pulse width T _p of the pulse laser light L is instructed by a not-shown input device, the pulse width T _p of the pulse laser light L, the detector 13a, It is set larger than the minimum pulse width at which the fluorescence intensity detected by 13b is maximized.

By increasing the pulse width T _p In this way, three-photon excitation effect occurring in the specimen A is greatly attenuated than the two-photon excitation effect. As a result, although the two-photon excitation fluorescence in the sample A to be measured is also attenuated, the unintended generation of the three-photon excitation fluorescence can be attenuated more greatly than the two-photon excitation fluorescence.

The regulation method of the pulse width T _p, will be described with reference to a specific example shown in FIG. 5 (c) from Fig. 5 (a). In each figure, the horizontal axis represents the pulse width of the pulsed laser light L, and the vertical axis represents the intensity of two-photon excitation fluorescence and three-photon excitation fluorescence detected by the detectors 13a and 13b.
5A shows the case where the intensity of the two-photon excitation fluorescence is larger than the intensity of the three-photon excitation fluorescence, and FIG. 5B shows the case where the intensity of the two-photon excitation fluorescence is smaller than the intensity of the three-photon excitation fluorescence. Yes. FIG. 5C shows a case where the maximum intensities of the two-photon excitation fluorescence and the three-photon excitation fluorescence are normalized as 1.

As shown in FIG. 5C, by widening the pulse width of the pulsed laser light L, the three-photon excitation fluorescence is attenuated more than the two-photon excitation fluorescence.
Therefore, the dispersion correction optical system 17 changes the pulse width of the pulsed laser light L to change the absolute value of the rate of change of the intensity of the three-photon excitation fluorescence, that is, the tangent of the quadratic function indicating the intensity of the three-photon excitation fluorescence. The pulse width of the pulsed laser light L is adjusted so that the absolute value of the inclination is not more than a predetermined threshold value.

  Specifically, for example, by setting the pulse width of the pulsed laser light L to be two to three times the minimum pulse width at which the fluorescence detected by the detector 13a or the detector 13b is maximized, The pulse width of the pulse laser beam L can be adjusted so that the intensity of the three-photon excitation fluorescence does not increase rapidly, and damage to the sample A due to the three-photon excitation fluorescence can be suppressed.

  As described above, according to the laser microscope 1 according to the present embodiment, since the fluorescence intensity of the three-photon excitation fluorescence can be measured simultaneously with the two-photon excitation fluorescence, the intensity is measured while measuring the fluorescence intensity of the two-photon excitation fluorescence. The above-described adjustment for widening the pulse width can be performed within a range in which the intensity sufficient for observation is maintained. Thereby, while observing the two-photon excitation fluorescence, the generation of the three-photon excitation fluorescence can be suppressed as much as possible, and the damage to the sample A by the three-photon excitation fluorescence is suppressed, and the sample A by the two-photon excitation fluorescence is suppressed. Observation can be performed with high accuracy. In addition, it can be confirmed how much the three-photon excitation fluorescence is actually suppressed.

  Further, the required amount of the fluorescence intensity of the two-photon excitation fluorescence and how much the three-photon excitation fluorescence needs to be suppressed vary depending on the type of observation and the type of sample. While measuring fluorescence and three-photon excitation fluorescence, it is possible to adjust the pulse width so that the balance of excitation efficiency is optimized.

  Further, by using the pulsed laser light L in which the excitation balance between the two-photon excitation fluorescence and the three-photon excitation fluorescence is adjusted as described above, for example, if the two-photon excitation stimulation for releasing the reagent applied to the sample A by two-photon excitation is performed. It is also possible to perform two-photon excitation stimulation in a state where the influence of the three-photon excitation effect is suppressed.

  Further, by providing the two detectors 13a and 13b, the two-photon excitation fluorescence and the three-photon excitation fluorescence generated in the sample A can be detected separately and simultaneously, and the pulse laser beam L by the dispersion correction optical system 17 is detected. The pulse width adjustment time can be shortened to reduce damage to the sample A.

  An operation unit (not shown) for operating the dispersion correction optical system 17 and an image generation unit (not shown) for generating respective images from the two-photon excitation fluorescence and the three-photon excitation fluorescence detected by the detectors 13a and 13b. And a display unit (not shown) for displaying the generated image. In this way, the dispersion correction optical system 17 can be operated by the operation unit while confirming the two-photon excitation fluorescence image and the three-photon excitation fluorescence image displayed on the display unit, and each image is desired. It is possible to adjust the pulse width of the pulsed laser light L so that the intensity becomes.

In the specific examples shown in FIGS. 5A to 5C, the dispersion correction optical system 17 changes the pulse width of the pulsed laser light L to change the absolute value of the rate of change of the intensity of the two-photon excitation fluorescence. That is, the pulse width of the pulsed laser light L may be adjusted so that the absolute value of the slope of the tangent of the quadratic function indicating the intensity of the two-photon excitation fluorescence is equal to or greater than a predetermined threshold.
By doing so, the pulse width of the pulsed laser light L can be adjusted by the dispersion correction optical system 17 so as to drastically improve the intensity of the two-photon excitation fluorescence, and the observation accuracy by the two-photon excitation fluorescence can be improved. It becomes possible to improve.

In the specific examples shown in FIGS. 5A to 5C, the dispersion correction optical system 17 changes the pulse width of the pulsed laser light L to change the three-photon excitation fluorescence with respect to the intensity of the two-photon excitation fluorescence. The pulse width of the pulsed laser light L may be adjusted so that the intensity ratio is equal to or less than a predetermined threshold value.
By doing so, the dispersion correction optical system 17 can adjust the pulse width of the pulsed laser light L based on the balance between the intensity of the two-photon excitation fluorescence and the intensity of the three-photon excitation fluorescence, which is suitable for observation. An increase in three-photon excitation fluorescence can be suppressed while ensuring the intensity of two-photon excitation fluorescence. This makes it possible to accurately observe the sample A by the two-photon excitation fluorescence while suppressing damage to the sample A by the three-photon excitation fluorescence.

[First Modification]
Below, the 1st modification of this embodiment is demonstrated with reference to FIG.
The laser microscope according to the first modification of the present embodiment includes an arithmetic device 101 and a memory 102 as shown in FIG. 6 in addition to the configuration of FIG. Specifically, while the adjustment of the dispersion correction optical system 17 is controlled from the arithmetic device (adjustment instruction means) 101, the arithmetic device 101 measures the intensity change of the two-photon excitation fluorescence and the three-photon excitation fluorescence, and the pulse width T _p The detected intensity change of each of the two-photon excitation fluorescence and the three-photon excitation fluorescence with respect to the change amount is correlated and recorded in the memory 102. At this time, the detected intensities of the two-photon excitation fluorescence and the three-photon excitation fluorescence are stored in the memory as values normalized so that the maximum intensity is 1.

  In this manner, a table in which the fluorescence detection intensities of two-photons and three-photons with respect to the adjustment amount of the pulse width are associated in the memory 102 is constructed. Then, conversely from the next time, the dispersion correction optical system 17 is controlled so that the detection intensities of the two-photon excitation fluorescence and the three-photon excitation fluorescence are in a predetermined range with respect to a value obtained by standardizing the maximum intensity as 1. To do.

  Specifically, for example, when the two-photon excitation fluorescence is set to 0.8 or more and the three-photon excitation fluorescence is 0.5 or less to the arithmetic device 101, the arithmetic device 101 corresponds from the table stored in the memory 102. The pulse width adjustment amount is read, and the pulse width of the pulse laser beam L is controlled so as to be the pulse width adjustment amount read by operating the dispersion correction optical system 17.

  In this way, for the purpose of performing the two-photon excitation fluorescence observation while suppressing the three-photon excitation fluorescence as much as possible and suppressing the two-photon excitation fluorescence as much as possible, the two-photon excitation fluorescence and the three-photon excitation fluorescence are used. If the intensity balance is set to a desired balance, the pulse width of the excitation pulse can be controlled so that it is realized automatically.

[Second Modification]
In the present embodiment, the method of adjusting the prism interval of the dispersion correction optical system 17 is adopted as the method of adjusting the pulse width. Instead, as shown in FIG. 7, the laser light source 2 and the beam shaping optics are used. For example, a dispersion adjusting system 26 such as two wedge-shaped glass plates is installed on the optical axis between the systems 20, and the overlapping state of the two sheets is adjusted as shown by arrows in FIG. Also good.

  By doing in this way, the optical path length d through which the pulse laser beam L passes through these glass plates is changed, the group velocity dispersion is intentionally given to the pulse laser beam L, and adjustment is performed so that the pulse width is widened. Can do. As a result, the longer the optical path length d passing through the glass plate, the greater the influence of group velocity dispersion and the wider the pulse width.

  In this case, in the method of adjusting the prism interval of the dispersion correction optical system 17, it is necessary to adjust the angle of the prism in addition to the prism interval, but the adjustment is simple because only the overlapping position of the glass plates is adjusted. There are advantages. Further, even in an apparatus configuration without the dispersion correction optical system 17, the pulse width can be adjusted.

[Third Modification]
Further, as a method of suppressing the generation of the three-photon excitation effect, a method of adjusting the average intensity of the pulse may be used. This can be realized, for example, by installing an ND filter on the optical axis of the pulsed laser light L between the laser light source 2 and the microscope body 3. In this method, the efficiency of the three-photon excitation effect can be easily adjusted by adjusting the transmittance of the ND filter.

[Fourth Modification]
In addition, as shown in FIG. 8, a detector 29 that detects three-photon excitation fluorescence may be separately provided on the transmission side of the sample A. Specifically, the pulsed laser light L transmitted through the sample A and the light mixed with the fluorescence from the sample are passed through the barrier filter 27 to transmit only the wavelength of the three-photon excitation fluorescence. The three-photon excitation fluorescence is condensed by the condenser lens 28 and detected by the detector 29.

  With this configuration, the number of optical elements between the sample A and the detector that detects the three-photon excitation fluorescence can be reduced. In addition, a detector can be installed near the sample A. Thereby, it is possible to improve the detection efficiency of the three-photon excitation fluorescence by preventing a loss at the time of transmission through the optical element of the originally weak three-photon excitation fluorescence and a decrease in the amount of light due to vignetting in the optical element.

  In the examples so far, one of the detectors 13a and 13b has a wavelength characteristic that separates the two-photon excitation fluorescence and the three-photon excitation fluorescence so that one of the detectors 13a and 13b can detect the three-photon excitation fluorescence. ing. Therefore, for example, when it is desired to perform 2Ch two-photon excitation fluorescence observation using both the photodetectors 13a and 13b, it is necessary to replace the excitation dichroic mirror 12 with one having a desired wavelength characteristic each time. However, if the three-photon excitation fluorescence is performed by the detector 29 on the transmission side, both the detectors 13a and 13b can be used for observation of two-photon excitation fluorescence. Therefore, the excitation dichroic mirror 12 is used for two-photon excitation observation from the beginning. There is also an advantage that the time and effort of replacement can be eliminated by installing it.

[Fifth Modification]
Further, in addition to a fluorescent reagent that generates two-photon excitation fluorescence in the sample A, a reagent that easily generates three-photon excitation fluorescence is used, so that the three-photon excitation fluorescence is intentionally induced. A method may be adopted in which the excitation efficiency of photon excitation fluorescence is improved and the efficiency of the three-photon excitation effect is easily adjusted.

[Sixth Modification]
Further, the excitation dichroic mirror 12 may be eliminated and only the detector 13a (or the detector 13b) may be provided. In this case, after adjusting the excitation efficiency of the three-photon excitation effect using the barrier filter 15 having the characteristic of transmitting only the three-photon excitation fluorescence wavelength, the barrier filter 15 is replaced with the characteristic of transmitting only the two-photon excitation fluorescence wavelength. Then, the two-photon excitation fluorescence is observed with the detector 13. Such a configuration has the advantage that less detectors and optical elements are required.

[Second Embodiment]
Next, a laser microscope according to the second embodiment of the present invention will be described with reference to FIG.
The laser microscope 1 according to the present embodiment is different from the first embodiment in that fluorescence obtained by combining two-photon excitation fluorescence and three-photon excitation fluorescence is detected by a single detector. Hereinafter, with respect to the laser microscope 1 of the present embodiment, description of points that are common to the first embodiment will be omitted, and different points will be mainly described.

  As shown in FIG. 9, the laser microscope 1 according to the present embodiment includes a single detector 13 instead of the detectors 13a and 13b in FIG. The barrier filter 15 transmits two-photon excitation fluorescence and three-photon excitation fluorescence and detects them without separating them by the detector 13.

Here, as described above, when the characteristics of the pulsed laser light L are changed, the tendency of the change in the excitation efficiency between the two-photon excitation fluorescence and the three-photon excitation fluorescence is different.
For example, the case of changing the pulse width T _p, 2-photon excitation fluorescence to the change of the pulse width varies in inverse proportion, three-photon excitation fluorescence varies inversely with the square of the pulse width. Therefore, if the two-photon excitation fluorescence and the three-photon excitation fluorescence are detected in a mixed state, the detected intensity is detected as a sum of an inversely proportional change and a square inversely proportional change.

  Therefore, when the pulse width is changed by changing the prism interval of the dispersion correction optical system 17, if the light detected by the detector 13 is predominantly two-photon excitation fluorescence dominant than the three-photon excitation fluorescence. The detected intensity changes in inverse proportion to the pulse width. On the other hand, when the three-photon excitation fluorescence is mixed to some extent in the light detected by the detector 13, the detection intensity changes as the sum of the inverse proportion of the pulse width and the inverse proportion of the square of the pulse width. .

When the average intensity P _ave is changed, the two-photon excitation fluorescence changes in a quadratic function with respect to the change in the average intensity, but the three-photon excitation fluorescence changes in a cubic function. Therefore, if the two-photon excitation fluorescence is overwhelmingly dominant over the three-photon excitation fluorescence, the detected intensity changes in a quadratic function with respect to the change in average intensity, and the three-photon excitation fluorescence is mixed to some extent. If detected, the detected intensity varies depending on the sum of the quadratic function and the cubic function.

  If the detection intensity of the detector 13 is observed as a function of the amount of change in pulse characteristics, as shown in FIG. 10, using such a tendency of change in detection intensity, the fluorescence of the two-photon excitation fluorescence in the sample A On the other hand, it can be determined whether the three-photon excitation fluorescence is in a very small state, or whether the three-photon excitation fluorescence has a certain intensity. Here, FIG. 10 shows changes in detection intensity and fitting curve of two-photon excitation fluorescence and three-photon excitation fluorescence with respect to the pulse width.

  When the pulse width is changed, it may be determined whether the change in the detection intensity is inversely proportional or the sum of the inversely proportional and the inversely proportional square. On the other hand, when the average intensity is changed, it may be determined whether the detected intensity changes in a quadratic function or the addition of a quadratic function and a cubic function. As described in the embodiment, it is preferable to change the pulse width in order not to sacrifice the feature of observation to the deep part of the specimen. Therefore, the pulse width is also adjusted in this embodiment.

  In this way, while measuring two-photon excitation fluorescence and three-photon excitation fluorescence simultaneously with the same detector, the change in fluorescence detection intensity against the amount of change in pulse width is plotted, and the change changes linearly with the change in pulse width. In this region, the three-photon excitation fluorescence is negligibly small compared to the two-photon excitation fluorescence. Therefore, the excitation efficiency of the two-photon excitation fluorescence is ensured as much as possible by adjusting the pulse width so that the detection intensity of the detector 13 becomes a boundary point between the linearly changing region and the non-linearly changing region with respect to the pulse width change. In this state, generation of three-photon excitation fluorescence can be suppressed.

  Specifically, the prism interval of the dispersion correction optical system 17 is adjusted by, for example, an electric stage, and information on the operation amount of this stage is input to the external arithmetic unit 30. Further, the detection intensity of the two-photon excitation fluorescence + three-photon excitation fluorescence detected by the detector 13 is also input to the arithmetic unit 30. The arithmetic unit 30 stores the detection intensity of the detector 13 with respect to the input movement amount of the stage. In this way, the intensity of two-photon excitation fluorescence + three-photon excitation fluorescence can be handled as a function with the pulse width of the pulsed laser light L as a variable.

  At this time, a boundary point between the region where the fluorescence intensity changes linearly with respect to the change in the pulse width and the region where the fluorescence intensity changes nonlinearly is calculated, and the prism of the dispersion correction optical system 17 is set so as to obtain the pulse width at that time If the interval is controlled and adjusted, the generation of the three-photon excitation fluorescence can be suppressed while the intensity of the two-photon excitation fluorescence is secured as much as possible. After this state, if the barrier filter 15 is replaced with one that transmits only two-photon excitation fluorescence, the detector 13 can observe the two-photon excitation fluorescence from the sample A in a state in which the generation of the three-photon excitation fluorescence is suppressed. It can be carried out.

  In reality, as shown in FIG. 10, it may be difficult to specify the position (boundary point) where the detection intensity of the two-photon excitation fluorescence and the three-photon excitation fluorescence is separated from the fitting curve. In this case, processing may be performed in which the detection intensity of the two-photon excitation fluorescence and the three-photon excitation fluorescence and the fitting curve are regarded as a boundary at a certain distance or more.

[First Modification]
A first modification of the present embodiment will be described below.
In this embodiment, the barrier filter 15 used is one that transmits two-photon excitation fluorescence and three-photon excitation fluorescence, but instead of this, as a removable barrier filter that transmits only two-photon excitation fluorescence. Only the intensity of two-photon excitation fluorescence may be detected.

  Since the intensity of the three-photon excitation fluorescence is very small, the change in the intensity of the two-photon excitation fluorescence when the pulse width is changed is compared with the change in the intensity of the two-photon excitation fluorescence + the three-photon excitation fluorescence using the barrier filter 15 By doing so, it is possible to easily detect the adjustment value of the pulse width from which the three-photon excitation fluorescence starts to increase.

[Second Modification]
Further, instead of adjusting the prism interval of the dispersion correction optical system 17 in order to change the pulse width, a combination of wedge-shaped glass plates as shown in FIG. 6 is used for the pulse light source 2 and the beam shaping optical system 20. A method of adjusting the pulse width by a method of changing the amount of dispersion applied to the pulses by adjusting the overlapping state of the glass plates by setting them on the optical axis between them may be adopted. In this case, in addition to simple adjustment of the pulse width, it is possible to adjust the excitation efficiency of the three-photon excitation fluorescence even in a configuration in which the dispersion correction optical system 17 is not present.

[Third Modification]
In addition, in order to suppress the efficiency of the three-photon excitation fluorescence, instead of changing the pulse width, the average intensity of the pulse is changed, and the change tendency of the two-photon excitation fluorescence + the three-photon excitation fluorescence at that time is a quadratic function. Alternatively, it may be determined whether the quadratic function and the cubic function are added and adjusted so that the three-photon excitation fluorescence is suppressed.

[Fourth Modification]
Further, a method may be adopted in which a reagent that easily generates three-photon excitation fluorescence is applied to the sample A to facilitate detection of the three-photon excitation fluorescence. This method has an advantage that it is easy to adjust the pulse so that the three-photon excitation fluorescence is suppressed by improving the detection accuracy of the weak three-photon excitation fluorescence.

A Sample 1 Laser microscope 2 Laser light source 3 Fluorescence microscope main body 4 Introduction optical system 5 Galvano mirror 8 Objective lens 9 Excitation dichroic mirror (branching means)
12 Fluorescent branching dichroic mirrors 13, 13a, 13b Detector (detection means)
17 Dispersion compensation optical system (pulse width adjusting means)
DESCRIPTION OF SYMBOLS 18 Alignment adjustment apparatus 19 Acousto-optic apparatus 20 Beam shaping optical system 21 Microscope incident correction apparatus 26 Dispersion adjustment system 29 Detector (detection means)
30 arithmetic device 101 arithmetic device 102 memory

Claims (6)

A laser light source for emitting pulsed laser light;
An objective lens for condensing fluorescence generated in the specimen while irradiating the specimen with pulsed laser light from the laser light source;
Detection means for detecting fluorescence collected by the objective lens;
Adjustment instruction means for instructing adjustment of the pulse width of the pulse laser beam;
When adjustment of the pulse width of the pulse laser light is instructed by the adjustment instruction means, the pulse width of the pulse laser light is set larger than the minimum pulse width at which the fluorescence intensity detected by the detection means is maximized A laser microscope comprising: Branching means for branching fluorescence collected by the objective lens into two-photon excitation fluorescence and three-photon excitation fluorescence;
The said detection means has a two-photon fluorescence detection part which detects the two-photon excitation fluorescence branched by the said branch means, and a three-photon fluorescence detection part which detects the three-photon excitation fluorescence branched by the said branch means. The laser microscope according to 1.   The pulse width adjusting means adjusts the pulse width of the pulse laser beam so that the absolute value of the rate of change of the intensity of the three-photon excitation fluorescence detected by the three-photon fluorescence detector is not more than a predetermined threshold value. Item 3. The laser microscope according to Item 2.   The pulse width adjusting means adjusts the pulse width of the pulse laser beam so that the absolute value of the rate of change of the intensity of the two-photon excitation fluorescence detected by the two-photon fluorescence detector is equal to or greater than a predetermined threshold. Item 3. The laser microscope according to Item 2.   The pulse width adjusting means is configured such that the ratio of the intensity of the three-photon excitation fluorescence detected by the three-photon fluorescence detection unit to the intensity of the two-photon excitation fluorescence detected by the two-photon fluorescence detection unit is equal to or less than a predetermined threshold. The laser microscope according to claim 2, wherein a pulse width of the pulse laser beam is adjusted. An operation unit for operating the pulse width adjusting means;
An image generation unit for generating respective images from the two-photon excitation fluorescence detected by the two-photon fluorescence detection unit and the three-photon excitation fluorescence detected by the three-photon fluorescence detection unit;
The laser microscope according to any one of claims 2 to 5, further comprising a display unit that displays an image generated by the image generation unit.

Images