JP4647641B2 - Laser microscope - Google Patents

Description

  The present invention relates to a laser microscope, and more particularly to a laser microscope suitable for an SHG (Second Harmonic Generation) microscope that detects a second harmonic of a sample.

  Various types of laser microscopes that utilize laser light have been developed according to their applications. A laser microscope collects laser light output from a laser onto a sample and receives reflected light, emitted light, or the like from the sample, thereby enabling observation or inspection of the sample.

  A confocal microscope is known as one embodiment of a laser microscope. The confocal microscope is attracting attention because it has excellent resolution and can acquire three-dimensional information of a sample. As one of such confocal microscopes, there is known a confocal microscope that scans irradiation light on a sample using a rotating pinhole substrate (see, for example, Patent Document 1). Light from the light source enters a pinhole substrate on which a plurality of rotating pinholes are formed. The light divided by the pinhole scans on the sample. In order to prevent uneven brightness on the sample, the pinholes are arranged on the substrate according to a spiral arrangement, and are arranged so that the pitches are equal in the radial direction and the circumferential direction along the spiral track.

  Alternatively, a confocal optical scanner in which pinholes are arranged so that the pinhole arrangement density is constant is known (see, for example, Patent Document 2). A micro lens can be used for the pinhole. However, the pinhole substrate according to these arrangements sometimes fails to obtain sufficient results in terms of suppressing uneven brightness and increasing the brightness of illumination on the sample. In addition, sufficient studies have not been made so far on a method for effectively designing such an array substrate.

  As another laser microscope, a second harmonic microscope that measures various physical properties of a sample by using second harmonics generated by irradiating the sample with laser light has been put into practical use. . The measurement using the second harmonic microscope is performed by focusing the laser beam output from the laser light source on the sample, scanning the sample, and detecting the second harmonic emitted from the inside of the sample. . The second harmonic microscope is attracting attention as an effective means for detecting structures and functions at the cell and protein level, particularly in the medical and biotechnology fields.

  When the sample is irradiated with light in this microscope, in many cases, multiphoton excitation fluorescence is generated from the sample together with second harmonic emission. In order to detect the second harmonic, the conventional second harmonic microscope has blocked the multiphoton excitation fluorescence by an optical filter. For this reason, information on multiphoton excitation fluorescence has been lost by the filter. Alternatively, some optical filters may not be able to sufficiently block multiphoton excitation fluorescence.

JP-A-5-127090 JP-A-5-119262

  The present invention has been made in view of the above-described prior art, and an object thereof is to provide a laser microscope that enables effective observation of a sample.

  A first aspect of the present invention uses a wavelength difference between a laser light source, a lens that collects light from the laser light source on a sample, a second harmonic generated from the sample, and multiphoton excitation light. A laser microscope comprising: means for separating; means for receiving the separated second harmonic for observation; and means for receiving the separated multiphoton excitation light for observation. Thereby, sample observation by the second harmonic can be performed effectively.

  In the laser microscope according to the first aspect, the means for separating using the wavelength difference is a dichroic mirror, and reflects one of the second harmonic of the light generated from the sample and the multiphoton excitation light. The other can be transmitted.

  The laser microscope according to the first aspect further includes an objective lens for collecting light from the sample, and the sample is disposed between the objective lens and a lens that focuses the sample. Is preferred. Thereby, a dichroic mirror for separating incident light and emitted light can be dispensed with.

In the laser microscope according to the first aspect, the means for receiving the second harmonic for observation and the means for receiving the multiphoton excitation light for observation each convert the received light into an electrical signal. Can be used.
Further, in the laser microscope according to the first aspect, means for further separating the second harmonic wave and the multiphoton excitation light separated by the means for separating using the wavelength difference using the generation timing difference. May be provided. Thereby, the sample observation by the second harmonic can be performed more effectively.

  The second aspect of the present invention uses a generation timing difference between a laser light source, a lens for condensing light from the laser light source on a sample, a second harmonic generated from the sample and multiphoton excitation light. And a means for receiving the separated second harmonic or multiphoton excitation light for observation for observation. Thereby, sample observation by the second harmonic can be performed effectively.

  In the laser microscope according to the second aspect, it is preferable that the means for separating includes a gate that passes through the second harmonic generated from the sample and opens and closes at a timing to block the multiphoton excitation light. Thereby, a 2nd harmonic and multiphoton excitation light can be isolate | separated effectively. Furthermore, it is preferable that the gate is opened for a predetermined time in synchronization with the pulse output timing of the laser light source, thereby transmitting the second harmonic and blocking the multiphoton excitation light. Thereby, timing control can be performed effectively. The gate can be synchronized with the pulse output timing of the laser light source with a predetermined delay. Thereby, timing control based on a sample and a microscope can be performed effectively.

  The laser microscope according to the first or second aspect preferably includes a pre-chirper that suppresses the spread of the pulse width due to dispersion of the optical system.

  ADVANTAGE OF THE INVENTION According to this invention, the laser microscope which enables the effective observation of a sample can be provided.

  Hereinafter, embodiments to which the present invention can be applied will be described. The following description is to describe the embodiment of the present invention, and the present invention is not limited to the following embodiment. For clarity of explanation, the following description is omitted and simplified as appropriate. Further, those skilled in the art will be able to easily change, add, and convert each element of the following embodiments within the scope of the present invention.

Embodiment 1 FIG.
FIG. 1 is a configuration diagram showing an outline of a laser microscope 100 in the present embodiment. The laser microscope 100 of this embodiment is an SHG (Second Harmonic Generation) microscope capable of detecting a second harmonic generated by a sample. The SHG microscope is an optical microscope that uses light of a predetermined wavelength to enter a sample and uses second harmonic generation (SHG). The SHG microscope can be used for observing various samples such as catalytic reaction surfaces, metal and semiconductor microstructures, etc., especially in the fields of medical and biotechnology, etc. It is an effective means. The image of the SHG light intensity represents the molecular orientation distribution and the like, and the deviation of the molecular concentration due to the non-equilibrium phenomenon and the pattern of the orientation distribution can be observed. A biological sample is composed of asymmetric biomolecules, and the SHG image is effective for extracting and observing the distribution of special structures in the biological body. In many cases, multiphoton excited fluorescence is generated from the sample along with second harmonic emission. The SHG microscope 100 of this embodiment can simultaneously observe multiphoton excitation fluorescence together with the second harmonic generated by the sample. Multiphoton excitation fluorescence is excitation fluorescence due to simultaneous absorption of two or more photons.

  In FIG. 1, 101 is a laser light source, 102 is a laser control unit that controls the laser light source, 103 is a pre-chirper that compensates for the expansion of chromatic dispersion by the optical system, 104 is a mirror, 105 is a beam expander, and 106 is a plurality of microlenses. 107 is a lens, 107 is a lens, 108 and 109 are mirrors, 110 is a first objective lens that collects light on the sample, 111 is a sample, and 112 is a first lens that collects light from the sample 111 2 objective lens, 113 is an optical filter that blocks light of a specific wavelength, 114 is a dichroic mirror, 115 and 116 are lenses, 117 and 118 are CCD (Charge Coupled Device) cameras that are examples of an imaging device, and 119 is image processing. It is a computer system which is an example of an apparatus. The light from the laser light source 101 enters the sample 111 via the optical system of FIG. 1, the second harmonic emitted from the sample 111 is detected by the CCD camera 117, and the multiphoton excitation fluorescence is detected by the CCD camera 118. It is detected separately.

  As the laser light source 101 of this embodiment, a laser device capable of generating second harmonic and multiphoton fluorescence is used. For example, an infrared light pulse using a mode-locked titanium-sapphire laser can be used for observation of biological cells. As the characteristics of the laser beam such as the laser beam wavelength, the laser beam intensity, the oscillation mode, the repetition frequency, and the pulse width, an appropriate one is selected depending on the sample and the observation method.

  The pulse laser beam output from the laser light source 101 enters the pre-chirper 103. The pre-chirper 103 compensates in advance for the phase shift of different frequency light in the laser light by the optical system. Thereby, the spread of the pulse width by the optical system of the pulse laser beam irradiated to the sample can be suppressed. The pre-chirper 103 will be described in detail later. The light that has been compensated for the phase shift by the pre-chirper 103 is reflected by the mirror 104 to change the optical path direction, and is incident on the beam expander 105. The beam spot diameter of the incident light is expanded by the beam expander 105.

  The beam having an enlarged spot diameter is incident on the microlens array disk 106. The microlens array disk 106 is rotated at a predetermined angular velocity by a driving device (not shown) such as a motor. The number of rotations is appropriately selected depending on the structure of the microlens array disk 106, the conditions of the imaging system, and the like. A typical rotational speed is approximately 60-30000 rpm. The microlens array disk 106 includes a plurality of microlenses arranged according to a predetermined rule, and can form a multifocal point on a sample. In addition, the focus on the sample moves as the microlens array disk 106 rotates, allowing many focused beams to scan the sample simultaneously. The structure of the microlens array disk 106 will be described in detail later.

  The light incident on the microlens array disk 106 is divided into a plurality of beams and incident on the lens 107. The plurality of incident beams are converted into parallel light by the lens 107 and enter the mirror 108. The light reflected by the mirror 108 and whose optical path direction is changed is changed again by the mirror 109 and is incident on the first objective lens 110.

  The first objective lens 110 condenses the incident multiple beams on the sample. As the objective lens 110 of this embodiment, a water immersion type objective lens is illustrated. The multi-beams divided by the microlens array disk 106 form multi-focal points on the sample 111 by the imaging action of the objective lens 110. The multifocal beam on the sample 111 is scanned on the sample 111 by the rotation of the microlens array disk 106.

  The sample 111 emits a second harmonic having a frequency twice that of the incident light and multiphoton excitation fluorescence by the incident light. A typical multiphoton excited fluorescence is two-photon excited fluorescence. The emitted light from the sample 111 is collected by the second objective lens 112. As the objective lens 112 of this embodiment, a water immersion type objective lens is illustrated. The second objective lens 112 is disposed on the side opposite to the first objective lens 110 with respect to the sample 111. The sample 111 is preferably disposed between the first objective lens 110 and the second objective lens 112, and the focal points of the two objective lenses are preferably substantially coincident on the sample. By receiving the emitted light from the sample not by the objective lens 110 that collects the incident light on the sample but by another objective lens 112 arranged in a transmission direction in which the output light of the laser passes through the sample, A dichroic mirror for separating emitted light can be dispensed with.

  FIG. 2 shows a typical relationship between the incident light on the sample, the second harmonic from the sample, and multiphoton excitation fluorescence. FIG. 2 shows an example, and the wavelength relationship of each light can vary greatly depending on the incident light and the sample. In FIG. 2, the X axis of the graph represents the light frequency, and the Y axis represents the light energy or intensity. 201 is a second harmonic emitted from the sample, 202 is multiphoton excitation fluorescence emitted from the sample, and 203 is incident light from the light source. The second harmonic 201 has a half wavelength (twice the frequency) of the incident light 203, and the wavelength of the multiphoton excitation fluorescence 202 is slightly larger than that of the second harmonic 201. The SHG microscope of this embodiment can detect each of the second harmonic 201 and the multiphoton excitation fluorescence 202 separately.

  The light from the objective lens 112 passes through the optical filter 113 and enters the dichroic mirror 114. The optical filter 113 blocks the transmitted light that has passed through the sample 111 from the laser light source 101. The optical filter 113 can be configured by a band pass filter or a low pass filter that cuts off the wavelength range of the output light 203 of the laser light source 101. Since the filter 113 blocks the incident light component of light from the sample 111, the light emission of the sample can be effectively observed.

  The second harmonic of the emitted light of the sample and the multiphoton excitation fluorescence are separated by the dichroic mirror 114. As shown in FIG. 2, there is a predetermined wavelength difference (frequency difference) between the second harmonic and the multiphoton excitation fluorescence. Therefore, when the dichroic mirror 114 is used, one light is reflected and the other light is reflected. These can be separated by permeation. The wavelength-reflectance (transmittance) characteristic of the dichroic mirror 114 is determined based on the second harmonic generated by the sample and the wavelength of multiphoton excitation fluorescence. Reflecting the wavelength range of the second harmonic and transmitting the wavelength range of the multiphoton excitation fluorescence, or transmitting the wavelength range of the second harmonic and reflecting the wavelength range of the multiphoton excitation fluorescence. it can.

  The dichroic mirror 114 of this example reflects the second harmonic and transmits multiphoton excitation fluorescence. The reflected second harmonic is collected by the lens 115 and enters the CCD camera 117. On the other hand, the transmitted multiphoton excitation fluorescence is condensed and incident on the CCD camera 118 via the lens 116. The CCD camera 117 preferably includes an image intensifier in order to effectively detect the second harmonic, which is weak light. The CCD cameras 117 and 118 detect incident light, convert it into a video signal, and transmit it to the image processing device 119. The image processing device 119 performs necessary image processing on the received detection data, and displays the captured image on the display.

  By separately detecting the second harmonic component and the multiphoton excitation fluorescence component of the emitted light, it is possible to effectively compare the respective image data. In addition, since the microscope of this embodiment receives light from the sample with the same objective lens, an accurate comparison is possible as compared with the case where a different objective lens is used. A CMOS sensor, a photodiode array, or the like can be used as the imaging device. Alternatively, an eyepiece device can be mounted instead of an imaging device such as a CCD camera. It is possible to construct a microscope without the use of a microlens array disk for the detection of second harmonics and multiphoton excitation fluorescence.

Embodiment 2. FIG.
FIG. 3 is a configuration diagram showing an outline of the SHG microscope in the present embodiment. 3, the same reference numerals as those in FIG. 1 denote the same elements, and the description thereof is omitted. In FIG. 3, reference numeral 301 denotes an intensified CCD camera which is an example of an imaging apparatus, and 302 denotes a delay circuit. The SHG microscope of this embodiment includes a mirror 306 instead of the dichroic mirror 114. Light from the sample 111 is reflected by the mirror 306, collected by the lens 115, and enters the CCD camera 301.

  The intensified CCD camera 301 of this embodiment is a gated intensified CCD camera that includes a gate that opens and closes at a predetermined timing. The intensified CCD camera 301 includes an image intensifier 303, a CCD 304, and a control / drive unit 305. The laser control unit 102 is connected in circuit with the intensified CCD camera 301 via the delay circuit unit 302. The laser control unit 102 transmits a clock signal synchronized with the repetition frequency of the laser to the intensified CCD camera 301 via the delay circuit unit 302.

  The image intensifier 303 has a gate function, and can block or amplify incident light at a predetermined timing. The image intensifier 303 of this embodiment controls the amplification of incident light based on the clock signal from the laser control unit 102. The image intensifier 303 can open and close the gate by changing the voltage applied to the microchannel plate inside the image intensifier 303. The control / drive unit 305 is a circuit unit that drives and controls the image intensifier 303 and the CCD 304 or controls the entire CCD camera 301. Since the gated intensified CCD camera is a well-known technique, detailed description thereof is omitted.

  FIG. 4 shows an example of a typical relationship between incident light output from the light source, second harmonic generated by the sample 111, and multiphoton excitation fluorescence. In FIG. 4, the X axis of each graph means time, and the Y axis means energy. 401 is the incident light that is output from the laser light source 101 and is incident on the sample, 402 is the second harmonic that the sample emits, 403 is multiphoton excitation fluorescence that the sample emits, and 404 is the timing when the gate of the CCD camera 301 is opened. Is shown. The second harmonic 402 and the multiphoton excitation fluorescence 403 are emitted at different timings. The second harmonic 402 is generated substantially simultaneously with the incident light. On the other hand, as shown in FIG. 4, the multiphoton excitation fluorescence 403 is emitted later than the second harmonic 402. The microscope according to this embodiment separates the second harmonic and the multiphoton excitation fluorescence using the difference in generation timing.

  Therefore, by setting the gate of the CCD camera 301 at a predetermined timing for a predetermined time, the multiphoton excitation fluorescence 403 can be blocked and the second harmonic 402 can be separated from the multiphoton excitation fluorescence 403 and detected. For example, in FIG. 4, the second harmonic 402 is detected by opening the gate at the incident timing of the laser beam, and closing the gate after a time corresponding to the emission time of the second harmonic 402. Subsequent multiphoton excitation fluorescence 403 can be blocked. Similarly, the multiphoton excitation fluorescence 403 can be separated from the second harmonic 402 and detected.

  The operation timing of the gate is determined based on the pulse frequency of the laser light source 101. The laser light source 101 outputs a pulse laser in accordance with the generated clock of the control circuit 102. The clock signal is delayed by a predetermined delay circuit and input to the CCD camera 301. The CCD camera 301 opens and closes the gate in synchronization with this clock signal. For example, the gate is opened according to the input clock signal, and the gate is opened only for a predetermined time. The timing delay process may be performed by the laser control unit or the CCD camera.

  The amount of delay is appropriately determined based on the optical system of the SHG microscope and the light emission characteristics of the sample, and may not be required by the system. The opening time of the gate is determined based on the emission time of the second harmonic and the emission time of the multiphoton excitation fluorescence. It is preferable to determine the opening time so that the second harmonic is received as much as possible and the multiphoton excitation fluorescence is received as little as possible. An appropriate timing and opening time can be determined by comparing the acquired image data while changing the timing delay amount and the gate opening time.

  By having the configuration of this embodiment, it is possible to effectively separate a strong multiphoton excitation fluorescence component that is difficult to be completely separated by an optical filter from the second harmonic component, and the S / N of SH image data. Can be improved. Various widely known ones can be used to control the temporal blocking or transmission of the emitted light and the detection, in addition to the gate of the image intensifier.

Embodiment 3 FIG.
FIG. 5 shows an example of the configuration of a microlens array disk 501 that can be used in the SHG microscope of the first or second embodiment. The microlens array disk 501 includes a plurality of microlenses Li that are examples of dots that transmit light. The arrangement position of each microlens Li on the disk 501 is specified by polar coordinates (ri, θi) with the rotation center of the disk as the origin. r i is the distance of the microlens Li from the center of rotation (hereinafter, the radial distance means this), and θ i is the relative angle of each lens. In FIG. 5A, reference numeral 502 denotes a microlens array composed of a plurality of microlenses Li, and each microlens is arranged in a region designated by 502 according to a predetermined rule. The microlens array 502 extends in a band shape in the circumferential direction of the microlens array disk 501.

  A dotted circle indicated by 503 indicates the spot diameter of the laser irradiated on the microlens array disk 501. From the viewpoint of efficiency, the diameter of the laser spot is preferably substantially the same as the radial distance between the lens farthest from the center of rotation and the closest lens. For example, when the distance from the radius center of the farthest lens is rl and the distance from the radius center of the nearest lens is r0, these radial distances are (rl-r0). For example, when the microlens array disk 501 makes one round, one scan of the entire laser light irradiation range on the sample is completed.

  FIG. 5 (b) shows an enlarged part of the microlens array. Here, the suffix i is given in the order in which r gradually increases (ri <ri + 1 <ri + 2). In FIG. 5B, the relationship (θi <θi + 1 <θi + 2) is established. Of course, the arrangement direction of the microlenses in the circumferential direction may be reversed so as to be arranged so that θ decreases. FIG. 5B shows microlenses arranged on the sth, (s + 1) th, and (s + 2) th rotations from the rotation center.

  As shown in FIG. 5A, when the microlens array disk 501 rotates, the beam separated by each microlens Li moves according to the rotation of the disk 501 and scans the sample. The imaging apparatus can obtain an image of a portion covered by the scanning line formed by each beam on the sample. High spatial resolution and temporal resolution can be realized simultaneously by scanning a wide range simultaneously with a plurality of beams within the scanning time.

  In the design method of the microlens array in this embodiment, the radial distance r from the rotation center of each microlens is determined so as to reduce the uneven brightness on the sample due to the plurality of divided beams. When one laser focal point scans, the light intensity per unit time and unit area (brightness of the scanning line) by the scanning line formed by the focal point is inversely proportional to the linear velocity of the laser focal point. Therefore, the brightness of each scanning line obtained by the plurality of divided beams is inversely proportional to the speed of each beam spot. Since the spot speed is faster as the coordinate r is larger, the scanning line of the lens having the largest coordinate r is the darkest, and the scanning line of the lens having the smallest coordinate r is the brightest. Therefore, in order to obtain uniform brightness on the sample, it is important that the number of scanning lines per area scanned on the sample increases from the center of rotation to the outside in the radial direction.

  The distance between adjacent scan lines formed on the sample by the microlens array 502 of this embodiment is designed to decrease as the brightness of each scan line decreases. In terms of lenses, the radial distance between the lenses with the closest radial distance (r) (the difference in radial distance between each lens (ri + 1−ri)) decreases as the radial distance r of the lens increases. Designed to. For example, in FIG. 5B, the relationship (ri + 2-ri + 1) <(ri + 1-ri) is established. For some lens pairs, the distance between the lenses in the radial direction can be substantially the same.

  The preferred design method for the radial distance of each microlens is the difference in the radial distance between the lenses with the closest radial distance, the brightness of the scan line formed by either lens or a lens that is close to the lens in radial distance. Decide on the basis. In particular, it is preferable that the radial distance is determined based on the brightness of one of the two lenses closest to each other or the brightness of both. Since the brightness of each scanning line is inversely proportional to the radial distance from the rotation center of the corresponding lens, the distance between adjacent lenses is preferably determined based on a fraction of the radial distance of one or both of the corresponding lenses. As a preferred example, the difference in radial distance can be determined to be proportional to the radial distance of one lens or the average of both radial distances. Below, an example of the preferable design method of the radial distance of each lens is shown.

ここで、ωは回転するレンズの角速度である。 The relationship between the linear velocity vi of the laser focus formed by the microlens Li on the disk and scanned by the rotation of the disk and the distance ri from the disk center of the lens forming the focus is expressed by the following equation.

Here, ω is the angular velocity of the rotating lens.

これを、ｒiについて解くと、次式が得られる。

ここで、ａはｒiの初項ｒ0と第２項ｒ1によって決定される定数であり、次式で与えられる。

初項ｒ0と第２項ｒ1は、走査線数や、ビーム・スポット径などに従って適切に決定することができる。
数式４によってｒiを求めることによって、各レンズの半径距離を決定することができる。 By making the distance between the scanning lines in the radial direction inversely proportional to the distance from the center of rotation, illumination with less unevenness can be realized. The distance from the center of rotation of the scan line can be determined by the following difference sequence.

Solving this for ri yields:

Here, a is a constant determined by the first term r0 and the second term r1 of ri, and is given by the following equation.

The first term r0 and the second term r1 can be appropriately determined according to the number of scanning lines, the beam spot diameter, and the like.
By obtaining ri according to Equation 4, the radial distance of each lens can be determined.

  In order to determine the position of each microlens on the disk, it is necessary to determine the coordinate θ in addition to the coordinate ri. In order to increase the light utilization efficiency, it is preferable to increase the radius of the lens as much as possible. Therefore, after determining the coordinates ri, it is important to determine the coordinates .theta.i of Li so as to increase the area occupied by the lens on the disk.

  For this purpose, it is preferable that the micro lens of the next periphery is arrange | positioned between the micro lenses adjacent to the circumference direction. Referring to FIG. 5B, the lens arranged on the (s + 1) th cycle is arranged between the adjacent lenses on the sth cycle. For example, it is preferable that the lens (Ln) adjacent in the radial direction is substantially disposed at the center position between the microlenses (Li, Li + 1) adjacent in the circumferential direction. Specifically, in FIG. 5B, (θi + θi + 1) / 2 and θn are substantially equal. One preferred method of determining θ is according to an conformal arrangement. In the equiangular arrangement, the angular coordinate difference between adjacent lenses in the circumferential direction is substantially the same between the lenses. For example, in FIG. 5B, the angle differences represented by (θi + 1−θi) are all equal.

ここでｎは自然数であり、最もレンズの半径を大きくできるように選ぶことが好ましい。等角配列において上式に従ってθ座標を決定することによって、θ座標に関してｓ周目の隣接レンズ間の中央に、ｓ+1周目のレンズを配置することができる。以上のように、各マイクロレンズの座標ｒiとθiとを決定することによって、効果的にレンズ配置設計を行うことができる。 Specifically, θ of each lens can be determined according to the following equation.

Here, n is a natural number and is preferably selected so that the radius of the lens can be maximized. By determining the θ coordinate according to the above equation in the equiangular arrangement, the lens of the (s + 1) th rotation can be arranged at the center between the adjacent lenses of the sth rotation with respect to the θ coordinate. As described above, the lens arrangement design can be effectively performed by determining the coordinates ri and θi of each microlens.

ｌは、螺旋における、隣接する周の間の距離である。これをθiについて解くと次式が得られる。

各マイクロレンズの座標ｒiは、各マイクロレンズによって形成される走査線の時間・面積あたり光強度が均一になるように決定することができる。例えば、上記の決定方法に従って、座標ｒiを決定することができる。θiを決定するために、螺旋の間隔ｌが決定されている必要がある。ディスク上のレンズ面積を大きくするためには、螺旋の間隔ｌは円周方向に隣接するレンズ間の最も小さい距離に基づいて決定することが好ましい。各走査線の明るさを均一にする配置を行う場合、ディスクの外側にあるほどレンズ間距離を小さくすることが好ましいので、螺旋の間隔ｌの好ましい決定方法の一つは、最も中心から離れたレンズＬlastと円周方向に隣接するレンズＬlast-1との間の距離に等しくなるように螺旋の間隔ｌを決定する。具体的には、ｌは次式によって表される。

上記設計方法によって、螺旋配列に従ってマイクロレンズを配置する場合に、レンズ面積を大きくするように効果的にレンズ配置設計を行うことができる。 Next, a method for designing a microlens array using a spiral array, which is another array that provides a dense lens arrangement, will be described. FIG. 6 shows a microlens array 601 arranged according to a helical arrangement. The microlenses Li are arranged on the solid line 602 in FIG. Specifically, the spiral arrangement is represented by the following formula.

l is the distance between adjacent perimeters in the helix. Solving this for θi yields:

The coordinates ri of each microlens can be determined so that the light intensity is uniform per time / area of the scanning line formed by each microlens. For example, the coordinate ri can be determined according to the above-described determination method. In order to determine θi, the helical interval l needs to be determined. In order to increase the lens area on the disk, it is preferable to determine the spiral interval l based on the smallest distance between adjacent lenses in the circumferential direction. When arranging the brightness of each scanning line to be uniform, it is preferable that the distance between the lenses is smaller as it is on the outer side of the disk. Therefore, one of the preferable methods for determining the helical interval l is the most distant from the center. The helical interval l is determined so as to be equal to the distance between the lens Llast and the circumferentially adjacent lens Llast-1. Specifically, l is represented by the following equation.

According to the above design method, when the microlenses are arranged according to the spiral arrangement, the lens arrangement design can be effectively performed so as to increase the lens area.

  FIG. 7 shows a configuration of a microlens array disk 701 according to another embodiment. In FIG. 7A, a microlens array disk 701 includes a band-shaped microlens array 702 extending in the circumferential direction. The microlens array 702 includes a plurality of microlenses Li, and each microlens Li is arranged according to a predetermined rule. The spot diameter 703 of the incident laser light is approximately the same as the width of the microlens array 702 from the viewpoint of efficiency.

  The microlens array 702 is composed of a plurality of sections Sj divided in the circumferential direction, and each section Sj has the same lens arrangement. One section passes through the laser spot 703 to complete a single scan on the sample. By rotating the microlens array disk 701 once, a plurality of scans corresponding to the number of sections can be performed. The number of sections in FIG. 7 is 8, and the number of scans per revolution is 8. Thereby, high-speed scanning becomes possible. Alternatively, since the number of rotations of the microlens array disk 701 can be reduced with respect to the same number of scans, disk shake can be suppressed, and uneven illumination can be reduced. By providing a plurality of sections in the microlens array, the lenses can be densely arranged by design as compared to the case of only a single section.

  FIG. 7B shows a part in one section. In FIG. 7B, the center of rotation exists in the lower part of the drawing. Each lens has a different radial distance r from the center of rotation, and the coordinate θ which is the position in the circumferential direction can be determined by, for example, an equiangular arrangement. The (s + 1) -th lens is disposed at the center position of θ adjacent to the s-th lens.

同様に、螺旋配列についても複数のセクションに分割することができる。螺旋配列における座標θijは、次式で与えることができる。

同じ走査線数、最大走査線間隔でマイクロレンズ・アレイを設計する場合、螺旋配列と等角配列とは、マイクロレンズ・アレイの半径に従って、選択することが好ましい。マイクロレンズ・アレイの最大半径（マイクロレンズの最も大きいｒi）に対する最小半径（マイクロレンズの最も小さいｒi）の比が所定値以上である場合には、等角配列によってレンズ配置設計を行うことが好ましく、所定値未満においては、螺旋配列によってレンズ配置設計を行うことが好ましい。螺旋配列は最大半径に対する最小半径の比に関係なく一定のレンズ密度になるのに対して、等角配列は、比を大きくすると密度が大きくなるからである。例えば、走査線数５７８本、走査線最大間隔１９．５μｍ、マイクロレンズ・アレイの最小半径４０ｍｍ、セクション数１２とした場合、ディスク上においてレンズの占める割合は、等角配列が螺旋配列よりも１％程度大きくすることができる。 The design of a microlens array with multiple sections can be performed as follows. The arrangement of the microlenses in each section can determine the coordinates rij according to [Equation 3], and can determine the coordinates θij according to the conformal arrangement. For the microlenses in each section Sj, coordinates rij are determined according to [Equation 3], for example. The coordinate θij of the lens Lij in the case of equiangular arrangement can be given by the following equation, where m is the number of scans per rotation of the disk.

Similarly, the helical array can be divided into a plurality of sections. The coordinate θij in the spiral arrangement can be given by the following equation.

When designing the microlens array with the same number of scanning lines and the maximum scanning line interval, it is preferable to select the spiral arrangement and the conformal arrangement according to the radius of the microlens array. When the ratio of the minimum radius (the smallest ri of the microlens) to the maximum radius (the largest ri of the microlens) of the microlens array is equal to or greater than a predetermined value, it is preferable to design the lens arrangement by an equiangular arrangement. If it is less than the predetermined value, it is preferable to design the lens arrangement by a spiral arrangement. This is because the spiral arrangement has a constant lens density regardless of the ratio of the minimum radius to the maximum radius, whereas the conformal arrangement increases in density as the ratio is increased. For example, assuming that the number of scanning lines is 578, the maximum scanning line interval is 19.5 μm, the minimum radius of the microlens array is 40 mm, and the number of sections is 12, the proportion of the lens on the disk is 1 in the conformal arrangement than in the spiral arrangement. % Can be increased.

  The microlens array disk can be applied not only to the SHG microscope but also to various types of laser microscopes, laser scanning devices such as inspection devices, and other optical systems. Further, the lens arrangement of this embodiment can be applied to the pinhole arrangement of the pinhole array in the confocal optical system. A pinhole is another example of a dot that transmits light.

Embodiment 4 FIG.
FIG. 8 shows the configuration of a pre-chirper applicable to the SHG microscope shown in the first or second embodiment. The pre-chirper can suppress the spread of the pulse width of the laser pulse by compensating in advance the phase shift caused by the frequency caused by the dispersion of the optical system. The pre-chirper of this embodiment has a great effect especially in a pulse laser of femtosecond order. Since the excitation efficiency of the second harmonic or multiphoton excitation fluorescence using a femtosecond pulse laser greatly depends on the pulse width, control of the pulse width is important. FIG. 8A conceptually shows how the pulsed light changes. By passing through the pre-chirper 800, the pulse width of the pulsed incident light is maintained even after passing through the next optical system 801 as in the waveform shown by the solid line. When the pre-chirper 800 is not used, the pulse width of the pulsed light is increased like a waveform indicated by a dotted line.

  As shown in FIG. 8B, the pre-chirper 800 of this embodiment is configured by four prisms 802-805. Incident light from the laser light source enters the vicinity of the apex angle of the first prism 802, refracts, and exits from the exit surface. Next, the light enters the vicinity of the apex angle of the second prism, and is refracted in the same manner and exits. The light incident from the fourth prism is sequentially incident on the prism, refracted, and emitted, and the light emitted from the fourth prism enters the subsequent optical system. Appropriate design conditions are selected for the material, refractive index, shape of each prism, and the arrangement conditions such as the distance and angle between the prisms. When the refractive index, first-order and second-order dispersion of each prism are determined, an appropriate pre-chirper can be designed by adjusting the inter-prism distance and the prism angle.

  The pre-chirper 800 cancels the group velocity dispersion in the optical system by causing negative group velocity dispersion. The pre-chirper 800 causes negative angular dispersion by utilizing the angular dispersion of the prism. A grating can also be used as an optical element that causes angular dispersion. However, it is preferable to use a prism from the viewpoint of light loss. By controlling the pulse width with the pre-chirper of this embodiment, effective observation can be performed.

It is a block diagram which shows the outline of the SHG laser microscope which concerns on embodiment of this invention. It is a figure which shows the wavelength-intensity characteristic of a laser incident light, a 2nd harmonic, and multiphoton excitation light in the SHG laser microscope which concerns on embodiment of this invention. It is a block diagram which shows the outline of the engagement SHG laser microscope in other embodiment of this invention. It is a figure which shows the time-intensity characteristic of a laser incident light, a 2nd harmonic, and multiphoton excitation light in the SHG laser microscope which concerns on other embodiment of this invention. 1 is a structural diagram showing an outline of a microlens array disk according to an embodiment of the present invention. 1 is a structural diagram showing an outline of a microlens array disk according to an embodiment of the present invention. 1 is a structural diagram showing an outline of a microlens array disk according to an embodiment of the present invention. It is a block diagram which shows the outline of the pre-chirper which concerns on embodiment of this invention.

Explanation of symbols

101 laser light source, 102 laser control unit, 103 pre-chirper, 104 mirror,
105 beam expander, 106 microlens array disk,
107 lens, 108, 109 mirror, 110 first objective lens, 111 sample,
112 second objective lens, 113 optical filter, 114 dichroic mirror,
115, 116 lens, 117, 118 CCD camera, 119 image processing device,
201 second harmonic, 202 multiphoton excitation fluorescence, 203 incident light, 301 CCD camera,
302 delay circuit, 303 image intensifier, 304 CCD,
305 control / drive unit, 306 mirror, 401 incident light, 402 second harmonic,
403 multiphoton excitation fluorescence, 404 gate opening timing,
501, 601, 701 microlens array disk,
502, 602, 702 microlens array,
503, 703 laser spot, 800 pre-chirper

Claims (10)

A laser light source;
A microlens array that splits the laser light from the laser light source to form a multifocal point on the sample;
Means for separating the second harmonic generated from the sample and the multiphoton excitation light using a generation timing difference;
Receiving the separated second harmonic or multiphoton excitation light for observation and converting it into an electrical signal, and
When receiving the second harmonic for observation, the separating means blocks the multiphoton excitation light that follows the second harmonic by a gate that opens in synchronization with the pulse output timing of the laser light source. Laser microscope. The laser microscope according to claim 1, wherein the gate is closed after a time corresponding to the generation time of the second harmonic. When the imaging means receives the multiphoton excitation light for observation, the separating means changes the timing at which the gate opens, thereby blocking the second harmonic,
  The laser microscope according to claim 1, wherein the imaging unit receives the multiphoton excitation light following the second harmonic for observation. The imaging means comprises a second harmonic imaging device that receives light to observe the second harmonic, and a multiphoton excitation light imaging device that receives light to observe the multiphoton excitation light.
The laser microscope according to claim 1, further comprising means for separating the second harmonic generated from the sample and the multiphoton excitation light using a wavelength difference. Means for separating by using the wavelength difference is a dichroic mirror, while transmitting the reflected other the of the second harmonic wave and multiphoton excitation light of the light generated from the sample, according to claim 4 Laser microscope. 6. The laser microscope according to claim 1, further comprising means for rotating the microlens array in order to scan the sample with a multifocal beam. The laser microscope according to any one of claims 1 to 6, further comprising a lens for condensing a plurality of beams from the microlens array on a sample. Furthermore, an objective lens for collecting light from the sample is provided,
The laser microscope according to claim 7 , wherein the sample is disposed between the objective lens and a lens that focuses the sample. The gate is synchronized with a pulse output timing and a predetermined delay of the laser light source, a laser microscope according to any one of claims 1 to 8.   Furthermore, the laser microscope of any one of Claim 1 thru | or 9 provided with the pre-chirper which suppresses the breadth of the pulse width by dispersion | distribution of an optical system.

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