JPH11119106A - Laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simultaneously perform fluorescent observation and the observation of the entire image of a sample in accordance with the sample and a purpose by detecting an interference signal generated by a laser beam reflected by a movable reflecting optical device and made incident on an optical path dividing element again, and a laser beam reflected by the sample and made incident on the optical path dividing element again. SOLUTION: The reflected light reflected inside the sample, and transmitted light transmitted through the sample 9, besides fluorescence are generated from the sample 9 irradiated with the laser beam. On the other hand, the part of the reflected light and the fluorescence are made incident on an objective lens 8 again, and reach a dichroic mirror 11a. The reflected light transmitted through the mirror 11a is made incident on an interference optical system 3, reflected by a beam splitter 3a, and synthesized coaxially with a reference beam reflected by a movable mirror 3b. At this time, the mirror 3b moves in an optical axis direction so as to align the length of the optical path of the reference beam with that of the reflected light, so that interference occurs between the synthesized reference beam and the reflected light, and the generated interference light is detected by an interference signal detecting optical system 4.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for focusing a near-infrared pulse laser on a sample surface and scanning the sample surface.
The present invention relates to a so-called multiphoton excitation laser scanning microscope that obtains an image by detecting fluorescence generated by excitation by multiphoton absorption including photon absorption. And a laser scanning microscope capable of performing reflection or transmission interference image observation simultaneously or by switching.

[0002]

2. Description of the Related Art One of the methods for observing a sample using an optical device such as a microscope is a method using a multiphoton excitation method.
The multiphoton excitation method is a method in which excitation usually performed by one photon (single photon) is performed by multiphotons. For example, in the two-photon excitation method, fluorescence excitation performed at a wavelength of 400 nm (single photon) is doubled. At a wavelength of 800 nm. At this time, 800
Since the energy of one photon at a wavelength of nm is half that of 400 nm, fluorescence excitation is performed using two photons at a wavelength of 800 nm.

A light source used for multiphoton excitation is, for example, a sub-picosecond pulse laser. This is because multiphoton excitation occurs with a probability that is almost proportional to the square of the photon density per unit volume and unit time.In a subpicosecond pulse laser, the pulse width is extremely narrow, so the probability of multiple photons being present is high. It depends. As described above, since fluorescence is generated only in a very small part of the sample by multiphoton excitation, an image with a very small depth of focus such as a so-called tomographic image can be obtained even with a thick sample.

However, since the laser light emitted from the pulse laser light source has a certain wavelength width, the pulse width of the laser light expands each time it passes through the optical element. Therefore, in a case where many optical elements exist before the laser beam reaches the sample, a problem arises in that the pulse width greatly expands at the sample position and multiphoton excitation does not occur. As a method of solving this problem, a method of pre-chirp compensation, in which a pulsed laser beam is used to emit short-wavelength light first using a prism pair, in other words, to delay long-wavelength light, is common. Is known to. Reference 1: "Femtoseco
nd pulse width control in microscopy by two-photon
absorption autocorrelation / by GJ Brakenhoff,
M. Muller & J. Squier / J. of Microscopy, Vol.179,
Pt.3, September 1995, pp.253-260 ": Details are given here, but are briefly explained here.

FIG. 8 shows an optical system for performing prechirp compensation. Here, among the four prisms, the first prism 31 is fixed, and the second prism 32, the third prism 33, and the fourth prism 34 are movable.

The second prism 32 is arranged on a holding table 35, and a driving device 36 for movement is provided on the holding table 35.
, And the driving unit 36 a is connected to the holding table 35. The holding table 35 is substantially orthogonal to the direction along the optical axis (not shown) connecting the first prism 31 and the second prism 32 as indicated by the arrow A and the arrow A as indicated by the arrow B. It has a moving mechanism that can move in the direction. As the moving mechanism, a fine movement mechanism that is conventionally used, such as a piezoelectric element, may be used. The moving mechanism of the third prism 33 and the fourth prism 34 is also configured in the same manner as the second prism 32, and is not illustrated here. Further, the adjustment direction is not limited to the direction shown in FIG.

[0007] A laser beam emitted from a pulse laser (not shown) enters a first prism 31. As mentioned above, a laser beam has a certain wavelength width,
The laser beam is decomposed for each wavelength on the incident surface 31 a of the prism 31, and spreads through the first prism 31 and proceeds. As the wavelength of light becomes shorter on the incident surface 31a, the laser beam is refracted more strongly. Therefore, the laser beam passing through the first prism 31 has a solid line (longer wavelength side) and a broken line (short wavelength side) as shown in FIG. Expands in the indicated range.

Next, the laser beam emitted from the first prism 31 enters the second prism 32. The laser beam incident on the second prism 32 travels different distances on the long wavelength side and the short wavelength side inside the prism, and is emitted from the emission surface 32a. The laser beam emitted from the emission surface 32a returns to a parallel light beam. At this time, the first prism 3
The distance between the first and second prisms 32 and the second prism 32
By changing the relative position with respect to its own optical axis, light with a shorter wavelength can have a shorter optical path length, and light with a longer wavelength can have a longer optical path length. That is, by using such an optical system, it is possible to realize a prechirp compensation in which “light of a short wavelength is emitted first and light of a long wavelength is delayed”.

Then, the laser beam emitted from the second prism 32 is applied to the third prism 33 and the fourth prism 3.
At this time, since the optical axis is moved with the movement of the second prism 32, the extension of the optical axis of the laser beam incident on the first prism 31 and the fourth The optical axes of the laser beams emitted from the prism 34 do not coincide. Therefore, the third prism 33 and the fourth prism 34 move so as to be bilaterally symmetric with respect to the arrangement positions of the first prism 31 and the moved second prism 32. As a result, even if the prism moves due to prechirp compensation, the fourth prism 3
The optical axis of the laser beam emitted from the laser beam 4 is kept constant. With such a configuration, the pulse width of a pulse laser light source having a wavelength width of about 10 nm or less can be adjusted in the conventional prechirp compensation.

[0010]

However, in general fluorescence observation including fluorescence observation by multiphoton excitation, fluorescence often occurs only in a specific part of the sample, so that the state of the entire sample is captured. There is a problem that is difficult. In particular, in the case of multiphoton excitation, the region where fluorescence is generated becomes extremely narrow, and it becomes more difficult to grasp the entire sample.

Further, in the conventional prechirp compensation optical system, since the range in which the pulse width can be adjusted is narrow, when a new optical system or an optical element is added to the optical path, the pulse width is increased by adding these optical systems. There was a problem that it was not possible to make adjustments to suppress the noise.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described problems. In a laser scanning fluorescence microscope capable of performing fluorescence observation by one-photon excitation and multi-photon excitation, fluorescence observation and the whole image of the specimen are performed according to the specimen and the purpose. It is an object of the present invention to provide a laser scanning microscope capable of simultaneously observing images.

[0013]

In order to achieve the above object, a laser scanning microscope according to the present invention comprises a pulse laser light source having a wavelength in the near infrared region and a laser beam from the pulse laser light source having a desired size. A collimating optical system that collimates the laser beam, an objective lens system that focuses the collimated laser light on the sample surface, scanning means that relatively two-dimensionally scans the collected laser light and the sample, In a laser scanning microscope provided with a fluorescence detection optical system for detecting generated fluorescence, the laser scanning microscope is disposed in an optical path between the pulse laser light source and the scanning unit, and reflects a part of the laser light from the pulse laser light source to a laser beam. An optical path splitting element for splitting the laser light so as to transmit the laser light, and an optical axis direction for reflecting one of the laser lights split by the optical path splitting element toward the optical path splitting element again. A possible movable reflective optical element, the laser light reflected by the movable reflective optical element and re-entering the optical path splitting element, and the remaining laser light split by the optical path splitting element and reflected by a sample and reflected by the sample; And an interference signal detecting optical system for detecting an interference signal generated by the laser light incident again on the optical disc.

With such a configuration, in addition to the fluorescence observation by multiphoton excitation including one photon by the laser scanning microscope, the state of the whole sample can be observed by the low coherence interference observation by the interference optical device. While observing the outline of the cell inside the cell with low coherence interference observation, it becomes possible to observe the fluorescence of a specific structure (chromosome or the like) to which a fluorescent dye is attached and detect a signal from a specific ion.

Further, another laser scanning microscope of the present invention is characterized in that it comprises a pulse width adjusting optical system for adjusting a pulse width of the pulse laser light source on the sample. In order to perform low coherence interference observation with high resolution, it is better that the laser wavelength width is large and the coherence length is short, but if the wavelength width is increased, the pulse width spread through the optical system is very large, and , The number of photons per unit time decreases, and multiphoton excitation does not occur. Therefore, by providing the pulse width adjusting optical system as in the present invention, the spread of the pulse width accompanying the optical system can be offset, so that the pulse width on the sample surface is reduced,
The photon density at which multiphoton excitation can sufficiently occur can be maintained.

Further, another laser scanning microscope of the present invention provides a full width at half maximum Δλ (n
m) is variable, and its variable range satisfies the following condition (1). By keeping the full width at half maximum of the oscillation wavelength in this range, low coherence interference observation and multiphoton excitation fluorescence observation with a resolution that does not hinder the observation can be performed even without the pulse width adjusting optical system. (1) 3 ≦ Δλ ≦ 100 Further, another laser scanning microscope of the present invention comprises at least one laser beam for condensing laser light from the optical path splitting element between the optical path splitting element and the movable reflective optical element. A fiber coupling optical system including a doublet, a single mode fiber for transmitting a focused laser beam, and a collimating optical system including at least one doublet for collimating the laser beam emitted from the single mode fiber are arranged. It is characterized by:

Therefore, even if the optical path between the optical path splitting element and the movable reflection optical element is lengthened, the single mode fiber can be bundled in a ring shape with the above-described configuration. The distance between the optical elements can be reduced, and the interference optical device can be made compact. Also, compared to a method in which an optical path bending mirror is arranged between the optical path splitting element and the movable reflective optical element, and the optical path is bent several times to make the interference optical device compact, high accuracy of surface accuracy and tilt adjustment accuracy is achieved. The required optical path bending mirror becomes unnecessary. Furthermore, since the spherical aberration and chromatic aberration are well corrected by using a doublet lens in the fiber coupling optical system and the collimating optical system, the wavefront of the reference light should be approximately the same as when no single mode fiber is used. Can be.

[0018]

FIG. 1 shows an embodiment of the present invention. FIG. 1 shows an optical device capable of fluorescence observation and low coherence interference observation. The pulse laser light source 1 is a light source for fluorescence observation and interference observation, and a laser beam emitted from the pulse laser light source 1 is enlarged to a desired size. The collimating optical system 2, the beam splitter 3 a as an optical path splitting element, the movable mirror 3 b as a movable reflecting optical element, and the light amount adjusting mechanism 3 constituting the interference optical system 3 for generating an interference image of the sample 9.
c, Condensing lens 4a, pinhole 4b, and photodetector 4 constituting interference signal detection optical system 4 for detecting an interference signal
c, a scanning optical system 5 which is a scanning means for scanning the sample 9 with a laser beam, a pupil projection lens 6 for guiding a laser beam from the scanning optical system 5 to an objective lens 8, an imaging lens 7, and a laser beam on the sample 9. The condenser lens 10a, the fluorescence cut filter 10b, the photodetector 10c, and the specimen 9 which constitute the transmitted light transmitted through the specimen 9 and the transmitted light transmitted through the specimen 9, and the transmitted light detection optical system 10 that detects and transmits the fluorescence. A dichroic mirror 11a, a condenser lens 11b, and a photodetector 11c, which constitute a fluorescence detection optical system 11 for detecting the generated fluorescence.
And an image processing device 12 for generating respective images from the outputs of the respective detection optical systems, and an image display device 13 for displaying the images.

The overall configuration of this optical device is as described above. When classified by function, the interference optical system 3 and the interference signal detection optical system 4 for detecting an interference signal constitute the main part of the low coherence interference device. , The scanning optical system 5, the fluorescence detecting optical system 11, and the transmitted light detecting optical system 10 constitute a main part of the laser scanning microscope. The pulse laser light source 1, the pupil projection lens 6, the imaging lens 7, and the objective lens 8
Is commonly used for both the low coherence interferometer and the laser scanning microscope.

The pulse laser light source 1 is a laser light source used for single-photon excitation or multi-photon excitation. In the case of multi-photon excitation, a sub-picosecond pulse laser is particularly suitable. The near infrared region is suitable as the wavelength region. The polarization state of the laser beam is desirably linearly polarized light. However, even if the polarization state is random polarization, linear polarization may be obtained in combination with a polarizing element.

Further, it is desirable that the pulse laser light source 1 has a variable full width at half maximum Δλ (nm) of the oscillation wavelength, and that the variable range satisfies the following condition (1). If the full width at half maximum of the oscillation wavelength is in this range, low coherence interference observation and multiphoton excitation fluorescence observation with a resolution that does not hinder observation can be performed. (1) 3 ≦ Δλ ≦ 100 Here, when the value is below the lower limit of 3 nm, the coherence length of the laser becomes too long, and the resolution of low coherence interference observation deteriorates. Conversely, when the upper limit of 100 nm is exceeded, the pulse laser light source 1 is used to generate multiphoton excitation.
The light intensity of the light must be very high, which will damage the specimen.

Further, when a pre-chirp compensation optical system is used and high-resolution low-coherence interference observation is emphasized, it is desirable that the following condition (1 ') is satisfied. (1 ′) 15 ≦ Δλ ≦ 100 Here, when the value is below the lower limit of 15 nm, the coherence length of the laser is long, and it becomes impossible to perform low-coherence interference observation with sufficiently high resolution. Conversely, when the upper limit of 100 nm is exceeded,
Even with the pre-chirp compensation optical system, the correction cannot be sufficiently performed, so that the light intensity of the pulse laser light source 1 must be very high in order to generate multiphoton excitation, and the specimen is damaged.

A light beam emitted from a pulse laser light source 1 for emitting a sub-picosecond monochromatic coherent light pulse having a wavelength in the near infrared region is collimated to a desired size by a collimating optical system 2 comprising an afocal optical system with two lenses. And enters the interference optical system 3. When the laser beam emitted from the laser light source is a divergent beam diverging from one point like a semiconductor laser, the collimating optical system 2
May be a single lens for collimating the divergent beam to a desired size or a so-called collimator combining a plurality of lenses.

The interference optical system 3 includes a beam splitter 3a as an optical path splitting element, a movable mirror 3b as a movable reflection optical element, and a light amount adjusting mechanism 3c. Interference optical system 3
Is split by the beam splitter 3a, a part of the incident laser beam is transmitted, and the remaining beam is reflected.

The laser beam reflected by the beam splitter 3a, that is, the reference light, is incident on a light amount adjusting mechanism 3c disposed forward in the traveling direction, and the light amount is adjusted. The reference light that has passed through the light amount adjustment mechanism 3c enters the movable mirror 3b. Since the mirror surface of the movable mirror 3b is arranged perpendicular to the optical axis, the reference light incident on the movable mirror 3b is reflected in a direction opposite to the incident direction. The position of the movable mirror 3b is moved in the direction of the optical axis by a moving mechanism (not shown) so that interference fringes generated by combining reflected light reflected from the specimen and reference light, which will be described later, are generated with good contrast. The reference light reflected by the movable mirror 3b passes through the beam splitter 3a and enters the interference signal detection optical system 4.

On the other hand, the laser beam transmitted through the beam splitter 3a transmits through the dichroic mirror 11a and enters the scanning optical system 5. The dichroic mirror 11a is one of the optical elements constituting the fluorescence detection optical system 11, and has a property of transmitting light having a wavelength in the near infrared region and reflecting light having a shorter wavelength, that is, fluorescence.

The scanning optical system 5 may be constituted by combining two not-shown light deflecting elements such as a galvanometer scanner and a polygon scanner in order to two-dimensionally scan a laser beam like raster scanning of a television. Since two-dimensional scanning can be performed by the deflecting element, at least one light deflecting element may be provided. When the scanning optical system 5 is not used, the same function as the scanning optical system 5 can be achieved by performing two-dimensional scanning of a stage (not shown) holding the specimen 9 as an alternative scanning unit.

The laser beam emitted from the scanning optical system 5 is focused by the pupil projection lens 6 on the image position I 'of the objective lens 8. At this time, it is desirable that the laser beam emitted from the pupil projection lens 7 be telecentric for low coherence interference observation. This is because, when the propagation of the pupil is considered on the wavefront, it is important for the interference observation that the light is emitted as a plane wave. For this purpose, it is necessary that the focal position of the imaging lens 7 on the objective lens 8 side coincides with the pupil position of the objective lens 8, but it is not so strict and ±
An error of about 5 mm is within a sufficiently allowable range.

Since the image position I 'is reduced and projected onto the sample 9 by the imaging lens 7 and the objective lens 8, the sample 9 is illuminated by two-dimensional scanning with a minute laser spot. The pupil position of the objective lens 8 and the optical deflecting element are conjugated by the pupil projection lens 6 and the imaging lens 7. Then, from the sample 9 irradiated with the laser, reflected light reflected inside the sample and transmitted light transmitted through the sample are generated, and when the sample is stained with a fluorescent dye, fluorescence is generated.

Among these, a part of the transmitted light and the fluorescence enters the transmitted light detection optical system 10. The transmitted light detection optical system 10 includes a condenser lens 10a, a fluorescence cut filter 10b, and a photodetector 10c. The transmitted light and the fluorescent light from the specimen 9 are collimated by the condenser lens 10a and enter the fluorescent cut filter 10b. The fluorescent cut filter 10b transmits the transmitted light having the same wavelength as the laser light source and has a wavelength shorter than the transmitted light. Since it has the property of reflecting or absorbing fluorescence, only transmitted light reaches 10c. In the photodetector 10c, an electric signal is generated according to the light intensity of the transmitted light, and the electric signal is sent to the image processing device 12.

On the other hand, a part of the reflected light and the fluorescence enters the objective lens 8 again, passes through the imaging lens 7, the pupil projection lens 6, and the scanning optical system 5, and reaches the dichroic mirror 11a. Here, in order to guide the fluorescence to the fluorescence detection optical system 11, the dichroic mirror 11a has a characteristic of transmitting reflected light having the same wavelength as the laser light source and reflecting fluorescence having a shorter wavelength than the reflected light. Therefore, only the fluorescence is reflected by the dichroic mirror 11a, and is collected by the light detector 11c by the condenser lens 11b. The photodetector 11c generates an electric signal according to the intensity of the fluorescent light, and the electric signal is sent to the image processing device 12. Note that the condenser lens 1
The confocal optical system or the non-confocal optical system can be selectively formed by making the pinhole 11d movable at the light condensing position 1b and inserting and removing the scanning optical system 5.

The reflected light transmitted through the dichroic mirror 11a enters the interference optical system 3, is reflected by the beam splitter 3a, and is coaxially combined with the reference light reflected by the movable mirror 3b. At this time, the movable mirror 3b moves in the optical axis direction so that the optical path length of the reference light matches the optical path length of the reflected light, so that interference occurs between the synthesized reference light and the reflected light, and the interference light causes the interference. The generated interference signal is detected by the interference signal detection optical system 4. In the low coherence interferometer composed of the interference optical system 3 and the interference signal detection optical system 4, the coherence length Lc of the pulse laser that determines the resolution is determined by the center wavelength λo of laser oscillation and its full width at half maximum Δλ. Is calculated. Lc = (λo) 2 / Δλ For example, if λo = 850 nm and Δλ = 30 nm, Lc is 2
When such a pulsed laser is used, the resolution in the optical axis direction is 25 μm or less, and only about 1/10 of that observed with good contrast provides an interference image with a substantial resolution of about 2.5 μm. Will be done.

The interference signal detecting optical system 4 includes a condenser lens 4
a, a pinhole 4b, and a photodetector 4c.
The interference light synthesized by the beam splitter 3a is condensed by the condensing lens 4a, but a pinhole 4b is disposed at the condensing position, and passes through the pinhole 4b and passes through the photodetector 4c.
Incident on. Since the interference signal detection optical system is a confocal optical system, it is possible to further improve the optical axis direction and the in-plane resolution of the low coherence interference observation and obtain a high-contrast observation image. The photodetector 4c generates an electric signal according to the light intensity of the interference light, and the electric signal is sent to the image processing device 12.

The electric signal sent from each photodetector to the image processing device 12 forms an image of the specimen 9 in synchronization with the scanning of the scanning optical system 5 and displays the image on the image display device 13. The images displayed on the image display device can be displayed simultaneously according to the situation, or can be switched and displayed.

As described above, since the present embodiment has an optical system capable of fluorescence observation and low coherence interference observation, fluorescence observation and low coherence interference observation by ordinary multiphoton excitation including one photon are performed. It can be carried out. Also, the scanning optical system is composed of a light beam deflecting means and a pupil projection lens, and the exit beam from the pupil projection lens is almost telecentric. Therefore, an optical system suitable for the interference optical system is configured.

In the embodiment of the present invention, the laser beam reflected by the beam splitter 3a is used for a low coherence interference optical device, and the transmitted laser beam is used for a laser scanning microscope. The reflected laser light may be used for a laser scanning microscope, and the transmitted laser light may be used for a low coherence interference optical device.

[0037]

First Embodiment FIG. 2 shows a first embodiment. This embodiment is an optical device that can simultaneously perform high-resolution low-coherence interference observation and two-photon excitation fluorescence observation. The same components as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted. Further, an image processing device and an image display device are omitted.

In this embodiment, the pulse laser light source 1 that emits a sub-picosecond coherent light pulse having a wavelength in the near infrared region is used as a pulse laser light source suitable for two-photon excitation. Emits a laser beam of 850 nm and Δλ (wavelength width) = 50 nm. As described above, in order to perform coherence interference observation at a high resolution, it is better that the laser wavelength width is large and the coherence length is short, but if the wavelength width is large, the pulse width due to passage through the optical system is extremely widened. And the number of photons per unit time on the sample surface decreases, and multiphoton excitation does not occur. Therefore, in this embodiment, a prechirp compensation optical system 14 composed of a prism pair is provided as a pulse width adjusting optical system so that a pulse width that causes multiphoton excitation is obtained on the sample.

In this embodiment, a prism having a small apex angle or a prism made of a glass material having a small Abbe number is used as the prism constituting the prechirp compensation optical system 14. If such a prism is used, a larger pulse width can be adjusted with a slight movement of the prism, so that an increase in the size of the prechirp compensation optical system can be prevented. Note that the apex angle of the prism is 45
° or less, the Abbe number of the glass material is more desirably 50 or less.

The laser beam emitted from the pulse laser light source 1 enters a prechirp compensation optical system 14 composed of a prism pair. Here, pre-chirp compensation is performed, and the laser beam is adjusted to have a pulse width that allows multiphoton excitation. The light beam that has exited the prechirp compensation optical system 14 is collimated to a desired size by the collimating optical system 2 and enters the beam splitter 3a having a division ratio of 1: 1. The laser beam reflected by the beam splitter 3a, that is, the reference light, is reflected by the reference mirror 3b via a rotary variable transmittance ND filter 15, which is a light amount adjustment mechanism, travels backward in the optical path and passes through the beam splitter 3a. The rotary transmittance variable ND filter 15 is formed, for example, by depositing a thin film whose transmittance changes stepwise in the circumferential direction on a disk-shaped glass substrate. Are used to obtain the optimum contrast of interference fringes with the same light intensity.

On the other hand, the laser beam transmitted through the beam splitter 3a passes through a scanning optical system 5, a pupil projection lens 6, an imaging lens 7, and a dichroic mirror 11a. Scan with spots. From the sample 9, fluorescence having a shorter wavelength than the laser light emitted from the sample 9 when excited by the laser light and reflected light having the same wavelength as the laser light reflected by the sample 9 are generated. Go. In this embodiment, the wavelength of the pulse laser light source 1 is 850 nm, and the wavelength of the fluorescence excited by two photons is 50 nm.
It is assumed to be about 0 to 600 nm. Therefore, the dichroic mirror 11a has a wavelength of 450 nm to 6 nm as shown in FIG.
It has a spectral characteristic that reflects light having a wavelength of 50 nm and transmits light having a wavelength of 700 nm or more.

Of the light from the sample 9, only the fluorescent light is reflected by the dichroic mirror 11a, and the condensing lens 11b
After the light is condensed, the light reaches the photodetector 11c and the fluorescence is detected. Although the dichroic mirror 11a reflects only the fluorescence, the dichroic mirror 1a actually reflects some laser light.
A laser cut filter 16 is provided between 1a and the fluorescence detection optical system 11c to prevent laser light from reaching the photodetector 11c.

In this embodiment, since the fluorescence is detected on the specimen side with respect to the scanning optical system 5, the position where the fluorescence is condensed by the condenser lens 11b moves in synchronization with the scanning at the position of the photodetector 11c. I do. Therefore, it is difficult to configure a so-called confocal optical system in which a pinhole is arranged at the light condensing position. However, since the excitation of the sample by the two-photon excitation method itself has the same effect as the confocal effect, the fluorescence detection 11
Can be obtained as in the present embodiment, the same effect as the confocal effect can be obtained.

The light reflected by the sample 9 passes through the dichroic mirror 11a and then enters the fluorescent cut filter 17. The fluorescent cut filter 17 removes a small amount of fluorescent light transmitted through the dichroic mirror 11a, and transmits only reflected light. The reflected light returns further in the optical path and reaches the beam splitter 3a. The reflected light is reflected by the beam splitter 3a and is coaxially synthesized with the reference light. Here, the position of the movable mirror 3b is adjusted so that the optical path length of the reflected light and the optical path length of the reference light are matched, and interference occurs. The interference light is condensed by the condensing lens 4a, and only the light transmitted through the minute confocal pinhole 4b placed at the condensing position is detected by the photodetector 4c.

In this embodiment, Δλ of the pulse laser light source 1
Is as large as 50 nm, so that the pulse width spread through the optical system is very large. Therefore, it becomes difficult to generate two-photon excitation when the sample is irradiated with the laser beam as it is. Therefore, in the present embodiment, the prechirp compensation optical system 14 is provided, and the spread of the pulse width caused by passing through the optical system is canceled by the prechirp compensation optical system 14 in advance, so that the pulse width of the sample 9 is reduced. I try to suppress the spread. In addition, the coherence length Lc of the laser 1 is about 14.5 μm, and it is possible to observe low coherence interference with sufficiently high resolution by itself.

In this embodiment, since the transmittance variable ND filter 15 is provided between the beam splitter and the reflecting mirror as a light amount adjusting mechanism, the light amount of the reference light and the light amount of the reflected light returning from the sample are measured. Can be balanced,
An interference image with higher contrast can be obtained. In addition, since the fluorescence detection optical path is provided with a laser cut filter and the interference image detection optical path is provided with a fluorescence cut filter, laser may enter the fluorescence detection optical path or fluorescence may enter the interference image detection optical path. And high contrast images can be obtained.

[0047]

Second Embodiment FIG. 4 shows a second embodiment. This embodiment has a configuration in which the optical device of the first embodiment is downsized. In the present embodiment, in the interference optical system,
Since only the configuration of the optical path of the reference light reflected by the beam splitter 3a is different from that of the first embodiment, only that portion is shown. In this embodiment, the same components as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted.

The laser beam reflected by the beam splitter 3a enters a light amount adjusting mechanism 20 including two linear polarizing plates 18 and 19. Of the two linear polarizing plates, the linear polarizing plate 18 disposed on the beam splitter 3a side has its polarization direction matched with the polarization direction of the laser beam. Also,
The one linear polarizing plate 19 is configured to rotate about the optical axis as a rotation axis by a rotation mechanism (not shown). Therefore, when the polarization direction of the linear polarizer 18 and the polarization direction of the linear polarizer 19 match each other (paranicol), the amount of transmitted light is maximum, and when the polarization directions are orthogonal (cross Nicol), the transmission amount is maximum. The light amount becomes almost zero. In a state other than paranicol or crossed nicol, the amount of transmitted light changes according to the polarization direction of the rotating linear polarizer 19. As described above, the light amount adjusting mechanism 20 can obtain an arbitrary transmittance, and can adjust the light amount of the reference light so that the contrast of the interference image is maximized.

The reference light whose light amount has been adjusted by the light amount adjusting mechanism 20 is condensed by the fiber coupling optical system 21 on the incident end face 22a of the single mode fiber 22.
The single mode fiber 22 emits laser light from the emission end face 22b of the single mode fiber 22 while maintaining the characteristics of the laser light at the time of incidence. The emitted reference light is collimated by the collimating optical system 23 and then collimated.
b.

In this embodiment, since a laser beam having a wavelength width is focused and collimated by the fiber coupling optical system 21 and the collimating optical system 23,
In these optical systems, it is necessary that chromatic aberration and spherical aberration are well corrected. Therefore, in the present embodiment, the fiber coupling optical system 21 and the collimating optical system 23
More preferably, the optical system includes at least one doublet.

As described above, in this embodiment, a single mode fiber is used for a so-called reference optical path having a mirror whose position in the optical axis direction is variable, which reflects the light beam reflected by the beam splitter 3a and reverses the optical path. Therefore, even if the optical path between the optical path splitting element and the movable reflective optical element is lengthened, the distance between the optical path splitting element and the movable reflective optical element can be shortened by bundling the single mode fibers in a ring shape, and interference optics can be achieved. The device can be made compact.

Further, compared with a method in which an optical path bending mirror is arranged between the optical path splitting element and the movable reflective optical element and the optical path is bent several times to make the interference optical device compact,
The need for an optical path bending mirror that requires high-precision surface accuracy and tilt adjustment accuracy is eliminated. Further, since the fiber coupling optical system 21 and the collimating optical system 23 are satisfactorily corrected for spherical aberration and chromatic aberration by using a doublet lens, the wavefront of the reference light is made to be substantially the same as when no single mode fiber is used. can do.

In the configuration of this embodiment, the optical path length OP (r) of the optical system from the fiber coupling optical system 21 to the collimating optical system 23, and the optical path length OP (o) from the beam splitter 3a to the sample surface 9 are set. Preferably satisfies the following condition (2). (2) 0.65 ≦ OP (r) / OP (o) ≦ 0.95 Here, when the value falls below the lower limit of 0.65, the amount of movement of the reflection mirror for adjusting the optical path lengths of the reference optical path and the observation optical path. When the value exceeds the upper limit of 0.95, the distance between the collimating optical system and the reflecting mirror becomes too short, which also makes the adjustment difficult. When this value exceeds 1, it goes without saying that it becomes impossible to match the optical path lengths of the reference optical path and the observation optical path.

More desirably, the following conditional expression should be satisfied to facilitate the adjustment. (2 ′) 0.7 ≦ O
P (r) / OP (o) ≦ 0.9 In this embodiment, O
The value of P (r) / OP (o) is about 0.85, so that the optical path lengths of the reference optical path and the observation optical path can be adjusted with high accuracy only by moving the reflecting mirror in the optical axis direction. Has become.

[0055]

Third Embodiment FIG. 5 shows a third embodiment. In this embodiment, high-resolution low-coherence interference observation and ordinary one-photon excitation fluorescence observation in which excitation light is in the near-infrared wavelength region (so-called I-photon excitation)
(R fluorescence observation). The same components as those in FIG.
Detailed description is omitted. Further, an image processing device and an image display device are omitted.

In this embodiment, as in the first embodiment, a pulse laser light source 1 that emits a sub-picosecond coherent light pulse having a wavelength in the near infrared region is used as a laser light source. A laser beam having a wavelength of 710 nm and Δλ (wavelength width) = 30 nm is emitted.

The light beam from the pulse laser light source 1 is collimated to a desired size by the collimating optical system 2. In the present embodiment, unlike the first embodiment, fluorescence observation by multiphoton excitation is not performed, so that the pulse width of the pulse laser light source 1 caused by the optical system does not matter. Therefore,
There is no pre-chirp compensation optical system between the pulse laser light source 1 and the collimating optical system 2. The laser light flux collimated to a desired size by the collimating optical system 2 is incident on a beam splitter 3a having a division ratio of 1: 1.
The laser beam reflected by the beam splitter 3a, that is, the reference light enters the light amount adjusting mechanism 27.

The light amount adjusting mechanism 27 of the present embodiment includes a liquid crystal 25
Is sandwiched between two linear polarizing plates 24 and 25. The linear polarizing plate 24 on the side of the beam splitter 3a is arranged so that its polarization direction matches the polarization direction of the laser 1, and the other linear polarization plate 25 has its polarization direction changed to the polarization direction of the linear polarization plate 24. Orthogonal to (cross nicol)
It is arranged to be in a state. Transparent electrodes (not shown) are provided on both surfaces of the liquid crystal 26, and a voltage is applied to the liquid crystal 26 via the transparent electrodes. When a voltage is applied to the liquid crystal 26 in such a configuration, the arrangement direction of the crystal molecules of the liquid crystal 26 changes, and the polarization state of light passing through the liquid crystal changes. Therefore, by adjusting the voltage applied to the liquid crystal 26, the light amount adjusting mechanism 27 is adjusted.
Thus, an arbitrary transmittance can be obtained, and the light amount of the reference light can be adjusted so that the contrast of the interference image is maximized.

On the other hand, the laser beam transmitted through the beam splitter 3a passes through the dichroic mirror 11a, the scanning optical system 5, the pupil projection lens 6, and the imaging lens 7, and is condensed by the objective lens 8, and the sample 9 Scan with spots. From the specimen 9, fluorescence having a longer wavelength than the laser light emitted from the specimen 9 excited by the laser light and reflected light having the same wavelength as the laser light reflected by the specimen 9 are generated. To go. In this embodiment, the wavelength of the pulse laser light source 1 is 710 nm, and the wavelength of the excited fluorescence is 760 nm.
nm to 800 nm. Therefore, as shown in FIG. 6, the dichroic mirror 11a has a spectral characteristic of reflecting light having a wavelength of 750 nm or more and transmitting light having a wavelength of 710 nm or less.

Although only the fluorescence is reflected by the dichroic mirror 11a and the laser light is transmitted, the laser light is removed by disposing a laser cut filter 22 just in case.
After the fluorescence transmitted through the laser cut filter 22 is collected by the condenser lens 11b, the fluorescence reaches the photodetector 11c to detect the fluorescence. In this embodiment, two-photon excitation is not performed as in the first embodiment, and therefore, in order to form a confocal optical system, fluorescence must be detected through a pinhole. Therefore, the fluorescence detection optical system 11 is disposed closer to the pulse laser light source 1 than the scanning optical system 5 is. With such an arrangement, the condensing position of the fluorescent light by the condensing lens 11b is always fixed at one point regardless of the scanning. Therefore, it is possible to configure a confocal optical system by disposing a pinhole at the condensing position. it can.

The light reflected by the sample 9 passes through the dichroic mirror 11a, enters the fluorescent cut filter 23, and transmits only the reflected light. The reflected light is reflected by the beam splitter 3a and is coaxially synthesized with the reference light. Here, the position of the movable mirror 3b is adjusted so that the optical path length of the reflected light and the optical path length of the reference light are matched, and interference occurs. The interference light is condensed by the condensing lens 4a, and only the light transmitted through the minute confocal pinhole 4b placed at the condensing position is detected by the photodetector 4c.

In the present embodiment, Δλ is large and the pulse width is widened by passing through the optical system. However, since one-photon excitation is assumed, the pulse width is not a problem. The coherence length Lc of the laser 1 is about 16.8 μm.
m alone, which enables low-coherence interference observation with sufficiently high resolution.

[0063]

Fourth Embodiment FIG. 7 shows a fourth embodiment. The present embodiment is an optical apparatus capable of simultaneously performing high-resolution low-coherence interference observation, two-photon excitation fluorescence observation, and differential interference observation of transmission by near-infrared light. The same components as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof will be omitted. Further, an image processing device and an image display device are omitted.

In this embodiment, a differential interference optical system is used to image the image of the sample with the laser light transmitted through the sample 9. The differential interference optical system is arranged such that the image position I ′ of the objective lens 8 is
Nomarski prism 28 disposed between the lens and the objective lens 8, and second Nomarski prism 1 disposed between the condenser lens 10a and the transmitted light detection optical system 10c.
9 and an analyzer 30. The position of the first Nomarski prism 28 may be anywhere between the objective lens image position and the objective lens in principle,
In consideration of the actual configuration, the shape of the Nomarski prism, and the like, it is desirable that they are arranged within 50 mm from the body of the objective lens.

Since the laser beam from the pulse laser light source 1 is linearly polarized, when it enters the first Nomarski prism 28, it becomes two slightly separated polarized beams. The two polarized light beams pass through the sample 9 and are condensed by the condenser lens 10a, enter the second Nomarski prism 19, and become one light beam again. Then, polarization interference occurs when the light passes through an analyzer 30 and is detected by the photodetector 10c as a so-called differential interference signal. The detected signal is displayed as an image on an image display device (not shown). In the present embodiment, the fluorescence cut filter 10b is provided to prevent the fluorescence from being mixed into the transmitted differential interference image, and the contrast of the differential interference image is improved.

In the case where the wavelength of the light source is infrared in the differential interference optical system as in this embodiment, the so-called near-infrared differential interference optical system (IR-DIC) is very useful for observation of a sample that is not too thick. It is valid. As described above, in this embodiment, since the optical system for detecting a so-called transmitted differential interference signal is provided, fluorescence observation by ordinary multiphoton excitation including one photon, low coherence interference observation, and differential interference observation are simultaneously performed. Or by switching.

It is desirable that the first and second Nomarski prisms 28 and 29 are configured to be insertable and removable from the optical path. With such a configuration, unnecessary optical elements can be removed from the optical path when differential transmission interference observation is not performed, so that a decrease in contrast can be prevented in fluorescence observation or low coherence interference observation.

Further, by setting the amount of share δ on the sample surface by the first Nomarski prism 28 and the objective lens to satisfy the following condition (3), the resolution in fluorescence observation can be sufficiently reduced. Differential interference observation with an effective differential interference effect can be performed. That is, when the intensity is less than 0.46 × λ / NA, the two spots are not separated from each other when considered by adding the intensities, and become one spot, so that the resolving power of the fluorescence observation hardly decreases. Furthermore, in the case of multiphoton excitation, fluorescence emission due to multiphoton excitation occurs only in a portion where two spots are superimposed, so that improvement in resolution can be expected. Also, even when performing differential interference observation of transmission at the same time,
By using image processing such as video enhancement without substantially lowering the resolution of fluorescence observation, a sufficient differential interference effect can be obtained. (3) 0.15 × λ / NA ≦ δ ≦ 0.46 × λ / NA Here, λ is the working wavelength, and NA is the numerical aperture of the objective lens. In conditional expression (3), the lower limit is 0.15 × λ / NA.
When the value is less than, the differential interference effect cannot be obtained even by using image processing such as video enhancement, and conversely, the upper limit of 0.46 × λ /
When the value exceeds NA, the two spots formed by the Nomarski prism and shared by the objective lens become two spots when the addition of the intensity is considered, and the resolving power of the fluorescence observation is reduced.

It is needless to say that any of the above embodiments is not limited to these configurations, but can be configured in various combinations.

[0070]

According to the present invention, a multiphoton-excited laser scanning microscope including one photon, a low coherence interferometer, an IR-
By combining an interference observation device such as a DIC,
For example, while observing the outline of cells inside tissue cells with low coherence interference observation or differential interference observation, it is possible to observe fluorescence of a specific structure such as a chromosome to which a fluorescent dye is attached and to detect signals from specific ions. A laser scanning microscope can be configured.

[Brief description of the drawings]

FIG. 1 shows a laser scanning microscope according to an embodiment of the present invention.

FIG. 2 is a diagram showing a laser scanning microscope according to the first embodiment of the present invention.

FIG. 3 is a diagram showing a spectral reflectance of a dichroic mirror used in the laser scanning microscope according to the first embodiment of the present invention.

FIG. 4 is a diagram showing an interference optical system in a laser scanning microscope according to a second embodiment of the present invention.

FIG. 5 is a diagram showing a laser scanning microscope according to a third embodiment of the present invention.

FIG. 6 is a diagram showing a spectral reflectance of a dichroic mirror used in a laser scanning microscope according to a third embodiment of the present invention.

FIG. 7 is a diagram showing a scanning laser microscope according to a fourth embodiment of the present invention.

FIG. 8 is a diagram showing a prechirp compensation optical system.

[Explanation of symbols]

 Reference Signs List 1 pulse laser light source 2, 23 collimating optical system 3 interference optical system 3a beam splitter 3b movable mirror 3c, 20, 27 light quantity adjusting mechanism 4 interference signal detecting optical system 4a, 11b condensing lens 4b, 11d pinhole 4c, 10c, 11c Photodetector 5 Scanning optical system 6 Pupil projection lens 7 Imaging lens 8 Objective lens 9 Sample 10 Transmitted light detection optical system 10a Condenser lens 10b Fluorescence cut filter 11 Fluorescence detection optical system 11a Dichroic mirror 12 Image processing device 13 Image display device 14 Prechirp compensation optical system 15 Variable transmittance ND filter 16 Laser cut filter 17 Fluorescence cut filter 18, 19, 24, 25 Linear polarizer 21 Fiber coupling optical system 22 Single mode fiber 22a Incident end face 22 Emission end surface 26 Liquid crystal 28 First Nomarski prism 29 Second Nomarski prism 30 Analyzer 31 First prism 31a First prism entrance surface 32 Second prism 32a Second prism exit surface 33 First Third prism 34 Fourth prism 35 Holder 36 Drive 36a Drive

Claims (16)

[Claims] 1. A pulse laser light source having a wavelength in a near-infrared region, a collimating optical system for collimating laser light from the pulse laser light source to a desired size, and a collimated laser light focused on a sample surface. In a laser scanning microscope including an objective lens system, scanning means for relatively two-dimensionally scanning the condensed laser light and the sample, and a fluorescence detection optical system for detecting fluorescence generated from the sample, An optical path splitting element disposed in an optical path between the laser light source and the scanning unit, for splitting the laser light from the pulse laser light source so as to reflect a part of the laser light and transmit the remaining laser light; A movable reflecting optical element movable in the optical axis direction for reflecting the one laser beam reflected toward the optical path splitting element again, and the optical path splitting element reflected by the movable reflecting optical element. Signal detection optical system for detecting an interference signal generated by the laser light again incident on the optical path splitter and the remaining laser light split by the optical path splitter and reflected by the sample and incident again on the optical path splitter A laser scanning microscope comprising an interference optical device having: 2. The apparatus according to claim 1, wherein a pulse width adjusting optical system for adjusting a pulse width of the pulse laser light source on the sample is provided on the sample side of the pulse laser light source. Laser scanning microscope. 3. A pulse width adjusting optical system comprising a prism pair or a grating pair or a mixture thereof, wherein the distance between the prism pair or the grating pair and the relative position with respect to the optical axis can be changed. 2. A laser scanning microscope according to claim 1. 4. A laser scanning microscope according to claim 1, wherein said interference optical system is a confocal optical system. 5. A laser scanning microscope according to claim 1, further comprising a light amount adjusting mechanism between said optical path splitting element and said movable reflecting optical element. 6. The laser scanning microscope according to claim 5, wherein said light amount adjusting mechanism is an ND filter capable of changing a transmitted light amount. 7. A laser scanning microscope according to claim 5, wherein said light amount adjusting mechanism comprises two linear polarizing elements, one of which is rotatable. 8. The laser scanning microscope according to claim 5, wherein said light amount adjusting mechanism comprises two linearly polarizing elements and a liquid crystal element sandwiched between said two linearly polarizing elements. 9. The method according to claim 1, wherein the full width at half maximum Δλ (nm) of the oscillation wavelength of the pulse laser light source is variable, and the variable range satisfies the following condition (1). Laser scanning microscope. (1) 3 ≦ Δλ ≦ 100 10. A fiber coupling optical system including at least one doublet for condensing laser light from the optical path splitting element between the optical path splitting element and the movable reflection optical element, and a condensed laser. 3. The laser scanning type according to claim 1, further comprising a single-mode fiber for transmitting light, and a collimating optical system including at least one doublet for collimating the laser light emitted from the single-mode fiber. microscope. 11. When the optical path length from the fiber coupling optical system to the collimating optical system is OP (r), and the optical path length from the optical path splitting element to the sample surface is OP (o), the following condition ( The laser scanning microscope according to claim 10, wherein 2) is satisfied. (2) 0.65 ≦ OP (r) / OP (o) ≦ 0.95 12. A first Nomarski prism disposed between the image position of the objective lens and the objective lens, a condenser lens disposed on the opposite side of the objective lens with respect to the sample, and a fluorescence cut filter. 3. The laser scanning microscope according to claim 1, further comprising an optical system having a second Nomarski prism and an analyzer for detecting a differential interference signal. 13. The laser scanning microscope according to claim 12, wherein the first Nomarski prism can be inserted into and removed from an optical path. 14. The laser scanning microscope according to claim 12, wherein the shear amount δ on the sample surface by the first Nomarski prism and the objective lens satisfies the following condition (3). (3) 0.15 × λ / NA ≦ δ ≦ 0.46 × λ / NA where λ is the wavelength of the laser light of the pulse laser light source,
NA is the numerical aperture of the objective lens. 15. The fluorescence detection optical system according to claim 1, further comprising a filter for cutting laser light, and the interference signal detection optical system including a filter for cutting fluorescence. Laser scanning microscope. 16. The scanning optical system according to claim 1, wherein said scanning optical system comprises a light beam deflecting means and a pupil projection lens, and an exit beam from said pupil projection lens is substantially telecentric. Laser scanning microscope.