JP2013134095A - Optical analyzer employing optical system of confocal microscope or multiphoton microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical analyzer employing an optical system of a confocal microscope or a multiphoton microscope which makes a moving speed of a position of a photodetection area higher than a speed obtained by rotating an optical element such as a mirror or a lens in an optical path behind an objective lens.SOLUTION: An optical analyzer 1 comprises: an optical element for optical path deflection which is rotationally moved to move a position of a photodetection area in a sample solution by changing a direction of an optical path of light incident/emitted from back of an objective lens 8; and photodetection area moving route displacement means which displaces, in a vibrational manner, the position of the photodetection area from a route of the position of the photodetection area formed by rotating the optical element for optical path deflection. The position of the photodetection area is displaced in the vibrational manner during movement along the route, thereby extending a scan length per unit time.

Description

  The present invention uses an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, and uses atoms, molecules or aggregates thereof dispersed or dissolved in a solution ( These are hereinafter referred to as “particles”), for example, biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, particulate objects such as viruses and cells, or non- It relates to optical analysis technology that can detect light from biological particles and obtain useful information in the analysis or analysis of those states (interaction, binding / dissociation state, etc.). More specifically, the present invention relates to an optical analyzer that enables various optical analyzes by detecting light from particles that emit light using the optical system as described above. In the present specification, a particle that emits light (hereinafter referred to as “luminescent particle”) is either a particle that emits light itself, or a particle to which an arbitrary luminescent label or luminescent probe is added. The light emitted from the luminescent particles may be fluorescence, phosphorescence, chemiluminescence, bioluminescence, scattered light, or the like.

  With the recent development of optical measurement technology, detection of weak light at the level of one photon or fluorescent single molecule using an optical system of a confocal microscope and an ultrasensitive photodetection technology capable of photon counting (one-photon detection)・ Measurement is possible. Thus, various photoanalysis techniques have been proposed for detecting characteristics of biomolecules, intermolecular interactions, or binding / dissociation reactions using such weak light measurement techniques. As such an optical analysis technique, for example, fluorescence correlation spectroscopy (FCS; see, for example, Patent Literature 1-3), fluorescence intensity distribution analysis (FIDA; for example, Patent Literature 4). ) And Photon Counting Histogram (PCH, for example, Patent Document 5) are known.

  Furthermore, the applicant of the present application is an optical analysis technique using an optical system capable of detecting light from a minute region in a solution such as an optical system of a confocal microscope or a multiphoton microscope in Patent Documents 6 to 8. Thus, a new optical analysis technique based on a principle different from optical analysis techniques such as FCS and FIDA has been proposed. In such a novel photoanalysis technique (hereinafter referred to as “scanning molecule counting method”), a micro area (hereinafter referred to as “light detection area”) that is a light detection area in the sample solution is used. In this case, the light detection region is dispersed in the sample solution while moving the position of the sample solution by the light detection region. When the randomly moving luminescent particles are included, the light emitted from the luminescent particles is individually detected, thereby detecting the luminescent particles in the sample solution one by one and counting the luminescent particles or in the sample solution. It is possible to obtain information on the concentration or number density of the luminescent particles.

JP-A-2005-09876 JP2008-292371 JP 2009-281831 A Patent No. 4023523 International Publication 2008-080417 International Publication No. 2011/108369 International Publication No. 2011/108370 International Publication No. 2011/108371 Japanese Patent No. 4391746

  In some of the optical analyzers using the optical system of the confocal microscope or the multiphoton microscope that executes the optical analysis techniques such as the scanning molecule counting method, FCS, FIDA, and PCH as described above, the inside of the sample solution is irradiated with light. A configuration for scanning by the detection region, that is, a configuration for moving the position of the light detection region of the objective lens is provided (Patent Document 9). In the configuration for moving the position of the light detection region, typically, the optical path behind the objective lens (excitation light source and / or mirror disposed in the optical path between the photodetector and the objective lens) is used. Alternatively, an optical element such as a lens is rotated to deflect the direction of light entering and exiting the rear of the objective lens, whereby the position of the light detection region formed in the sample solution in front of the objective lens is moved. In the case of movement of the position of the light detection region by the rotation of the optical element in the optical path behind the lens, the movement speed is determined by the rotation speed of the optical element, and therefore the rotation of the optical element cannot be increased indefinitely. The moving speed of the position of the light detection area is limited.

  However, at the time of carrying out the above optical analysis technique, the position of the light detection region is adjusted at a speed higher than the moving speed of the position of the light detection region obtained by setting the rotation speed of the optical element to an allowable maximum speed. Sometimes you want to perform a move. For example, in the scanning molecule counting method or FIDA, in order to obtain a good measurement result, it is necessary to detect a certain number of luminescent particles to be observed, so that it becomes an observation target in a sample solution. If the concentration of the luminescent particles is low, scanning within the sample solution over a longer distance will be performed. In such a case, the faster the moving speed of the position of the light detection region, the shorter the measurement time, which is advantageous.

  Thus, one object of the present invention is to provide an optical analyzer using a confocal microscope or an optical system of a multiphoton microscope for optical analysis techniques such as the above-described scanning molecule counting method, FCS, FIDA, and PCH. It is to provide a novel configuration in which the moving speed of the position of the light detection region can be made higher than the speed obtained by simply rotating the optical element in the optical path behind the objective lens.

  According to the present invention, the above-described problem is an optical analyzer that detects and analyzes light from a light detection region in a sample using an optical system of a confocal microscope or a multiphoton microscope. An optical path deflecting optical element that moves the position of the optical detection area by changing the direction of the optical path entering and exiting from the rear (image space side) of the objective lens, and an optical detection area formed by rotation of the optical path deflection optical element And a light detection region moving path displacing means for oscillatingly displacing the position of the light detection region from the position path. In such a configuration, the “light detection region” of the optical system of the confocal microscope or multiphoton microscope is a confocal volume, that is, a minute region in which light is detected in the microscope, and is excited from the objective lens. When light is given, it corresponds to the region where the excitation light is collected. The “optical path deflecting optical element” is typically a reflection mirror, but may be an optical element that can change the direction of incident or outgoing light by appropriately rotating a lens or the like. The sample is typically a light emitting particle (light emitting particle) such as an atom, molecule or aggregate thereof, and is not fixed to the substrate or the like, and freely moves in the solution in Brownian motion. It may be a solution in which particles are dispersed or dissolved. The luminescent particles are typically fluorescent particles, but may be other luminescent particles (such as phosphorescent particles).

  The apparatus of the present invention is basically directed to the direction of light rays entering and leaving the objective lens by rotating the optical element for deflecting the optical path, similarly to the apparatus described in the above-mentioned patent documents. Thus, the apparatus may have a configuration in which the position of the light detection region corresponding to the focal region on the front side (object space side) can be moved. However, in the case of the apparatus of the present invention, a mechanism (light) that further oscillates the position of the light detection region relative to the path of the position of the light detection region formed by the rotational movement of the optical element for deflecting the light path. Detection area moving path displacement means) is provided. According to such a configuration, the light detection area moves while oscillating and displacing along the path determined by the rotational motion during rotation of the optical element for deflecting the optical path. The distance that the detection region passes is longer than the length of the path determined by the rotational movement of the optical element for deflecting the optical path. That is, the actual moving speed of the light detection region is faster than the speed of following the path determined by the rotational movement of the optical path deflecting optical element. Therefore, if the rotational movement of the optical path deflecting optical element is maximized, A scanning speed exceeding the maximum speed when moving the light detection region along the path determined by the rotational movement of the optical element for deflecting the optical path can be achieved.

  In an embodiment of the above configuration, the optical element for deflecting an optical path typically has a rotation axis and a mirror surface extending in a direction inclined with respect to a plane perpendicular to the rotation axis. It may be a deflection mirror that is driven to rotate around. In this case, the light detection area moving path displacing means oscillates the direction of the mirror surface during rotation of the deflection mirror, and the position of the light detection area is oscillated from the path determined by the rotational movement of the optical element for deflecting the optical path. It may be configured to be displaced. As a mechanism for oscillating the direction of the mirror surface, in one embodiment, first, it extends in a plane perpendicular to the rotation axis along the outer periphery of the deflection mirror and rotates integrally with the deflection mirror. An annular member is provided, and a rod-like member extending in the diameter direction from the inner periphery of the annular member is connected to the deflection mirror. Means for generating a torsional vibration is provided about the extending direction of the rod-shaped member as an axis, and the mirror surface of the deflection mirror is rotated in the extending direction of the rod-shaped member by the torsional vibration of the rod-shaped member during the rotation of the deflecting mirror. The direction of the mirror surface may be oscillatingly displaced in such a manner that it rotates or oscillates oscillating around. As a means for generating the torsional vibration in the rod-shaped member, for example, a configuration in which a piezoelectric element that contacts the back surface of the deflection mirror and applies a vibrational rotational force around the extending axis of the rod-shaped member or an annular member is provided. A knurled groove formed on the outer periphery or back surface is formed, and a protruding member that contacts the knurled groove without rotating integrally with the annular member is provided, and the protruding member is knurled while the deflection mirror and the annular member are rotating. A structure in which mechanical vibration is generated by sequentially colliding with the continuous convex portions of the member, and thereby, torsional vibration of the rod-like member may be employed. Moreover, about a rod-shaped member, a member and a shape may be selected so that it may have arbitrary elasticity.

  According to the aspect in which the direction of the mirror surface is oscillatingly displaced, the light detection region moves along the orbit defined by the rotation of the deflecting mirror while oscillating and oscillating substantially in the radial direction of the orbit. Become.

  In another aspect of the light detection area moving path displacement means, at least one position of the lens in the optical system or the tip of the optical fiber emitting excitation light or the pinhole is oscillated in the optical axis direction. A means for displacing may be employed. In this case, the position of the light detection region is oscillatingly displaced in the direction of the optical axis from the path of the position of the light detection region formed by the rotation of the optical element for deflecting the optical path.

  The optical analysis technique executed by the apparatus of the present invention typically includes a scanning molecule counting method (Patent Documents 6-8), FIDA (Patent Document 4), FCS (Patent Documents 1-3), and the like. Used for analysis or analysis of the state in solution of biomolecules such as proteins, peptides, nucleic acids, lipids, sugar chains, amino acids or aggregates thereof, and particulate biological objects such as viruses and cells. May be used to analyze or analyze the state of non-biological particles (eg, atoms, molecules, micelles, metal colloids, etc.) in solution, and such cases are also within the scope of the present invention. Should be understood.

  Thus, according to the configuration of the present invention described above, the position of the light detection region is substantially perpendicular to the traveling direction of the path when moving along the path formed by the rotation of the optical element for deflecting the optical path. Since it is oscillatingly displaced in the direction of the path on the path plane or in the direction perpendicular to the path plane, the scanning distance per rotation of the optical element for deflecting the optical path increases. In other words, when the light detection area goes around a closed track, the movement distance of the position of the light detection area per cycle becomes longer, and therefore the movement speed of the position of the light detection area is simply used for optical path deflection. It becomes possible to make it faster than the speed by the rotation of the optical element. According to such a configuration, scanning over a longer distance is achieved in a shorter time, and the time required for optical measurement is reduced.

  Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

FIG. 1A is a schematic diagram of the internal structure of an optical analyzer that realizes the present invention. FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope). FIG. 1C is a schematic diagram of a mechanism for changing the direction of the mirror 7 to move the position of the light detection region in the sample solution. FIG. 2A is a schematic perspective view of one embodiment of a mirror and a mirror deflector for moving the position of the light detection region according to the present invention, and FIG. It is a top view of the site | part which supports the mirror of the mirror deflector seen from the back side of a mirror, FIG.2 (C) is sectional drawing of a mirror deflector from the direction of the line (C) in (B). FIG. 2D is a schematic plan view of a movement path followed by the position of the light detection region obtained by the mirror deflector of FIG. FIG. 3A is a cross-sectional view similar to FIG. 3C of the embodiment when the orientation of the piezoelectric element is changed in the mirror deflector of FIGS. FIG. 3B is a schematic perspective view of another embodiment of a mirror deflector that realizes the movement path illustrated in FIG. FIG. 4A shows an objective lens according to an embodiment in which a mechanism for oscillating the position of the light detection region from the path of the position of the light detection region formed by the rotation of the deflection mirror is provided in the objective lens. FIG. FIGS. 4B to 4D show the position of the light detection region oscillatingly displaced from the path of the position of the light detection region formed by the rotation of the deflection mirror in the introduction portion of the excitation light into the microscope. It is a schematic diagram of embodiment with which the mechanism is provided. (B) When the position of the collimating lens is oscillatingly displaced. (C) When the position of the pinhole is oscillatingly displaced. (D) When oscillatingly displacing the position of the exit end of the optical fiber. FIG. 2E is a schematic view of the movement path followed by the position of the light detection region achieved in the cases of FIGS. 4A to 4D as seen from the direction perpendicular to the optical axis.

1 ... Optical analyzer (confocal microscope)
DESCRIPTION OF SYMBOLS 2 ... Light source 3 ... Single mode optical fiber 3a ... Fiber exit end 4 ... Collimator lens 4a ... Pinhole 5 ... Dichroic mirror 6, 7, 11 ... Reflection mirror 7a ... Mirror deflector 8 ... Objective lens 8a ... Lens in objective lens Element 8b ... objective lens barrel 9 ... microplate 10 ... well (sample solution container)
DESCRIPTION OF SYMBOLS 12 ... Condenser lens 13 ... Pinhole 14 ... Barrier filter 15 ... Multi-mode optical fiber 16 ... Photo detector 17 ... Mirror deflector motor 17a ... Stage position change device 18 ... Computer 20 ... Ring member 22 ... Rod-shaped member 24 ... Mirror base DESCRIPTION OF SYMBOLS 30 ... Piezoelectric element 30a ... Drive member 32 ... Tongue part 34 ... Terminal electrode 34a ... Terminal 40 ... Knurled convex part 42 ... Projection member

  Hereinafter, preferred embodiments of the present invention will be described in detail.

Configuration of Optical Analysis Apparatus The present invention includes an optical system and a photodetector of a confocal microscope capable of executing a scanning molecule counting method, FCS, FIDA, PCH, etc. as schematically illustrated in FIG. It is applied to an optical analysis device formed by combining With reference to FIG. 1 (A), the optical analysis apparatus 1 is comprised from the optical systems 2-17, and the computer 18 for acquiring and analyzing data while controlling the action | operation of each part of an optical system. The optical system of the optical analyzer 1 may be the same as the optical system of a normal confocal microscope, in which the laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is a fiber. At the exit end, the light diverges at an angle determined by a specific NA, and is emitted as parallel light by a collimator (collimator lens) 4 and reflected by a dichroic mirror 5 and reflection mirrors 6 and 7. , And enters the objective lens 8. Above the objective lens 8, a microplate 9 in which sample containers or wells 10 into which a sample solution of 1 to several tens μL is dispensed is typically arranged is emitted from the objective lens 8. The laser beam is focused in the sample solution in the sample container or well 10 to form a region (focus region) having a high light intensity as schematically shown in FIG. The focal region is usually a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL, and is referred to as a confocal volume. In the confocal volume, the light intensity is typically a Gaussian or Lorentzian distribution with the center of the region at the apex, and the effective volume is bounded by the surface where the light intensity is 1 / e ^2. The volume of a substantially elliptic sphere. In the sample solution, luminescent particles that are observation objects, typically molecules to which a luminescent label such as a fluorescent dye is added are dispersed or dissolved, and when the luminescent particles enter the focal region, The luminescent particles are excited and light is emitted. The emitted light (Em) passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, is collected by the condenser lens 12, and an image of the focal region is formed by the pinhole 13. The pinhole 13 is disposed at a position conjugate with the focal position of the objective lens 8, whereby only light emitted from within the focal region as illustrated in FIG. 1B passes through the pinhole 13. Light from other than the focal plane is blocked.

  Thus, the light that has passed through the pinhole 13 passes through the barrier filter 14 (here, only the light component of a specific wavelength band is selected) and is introduced into the multimode optical fiber 15 and the corresponding photodetector. 16 is reached. In the photodetector 16, the sequentially arriving light is converted into a time-series electric signal and input to the computer 18, and processing for optical analysis is performed in a manner described later. As the photodetector 16, an ultrasensitive photodetector that can be used for photon counting is preferably used, so that light from one luminescent particle, for example, one or several fluorescent dye molecules can be used. The weak light can be detected. The photodetector 16 typically employs a photodiode, more preferably an APD (avalanche photodiode). Thus, with the above configuration, the intensity of light from the luminescent particles is measured.

  In the optical system of the above optical analyzer, the sample solution is scanned by the photodetection area by the photodetection area when the scanning molecule counting method is executed or depending on the mode of execution such as FCS, FIDA, and PCH. That is, the position of the focal region (that is, the light detection region) is moved in the sample solution. As a mechanism for moving the position of the light detection region, a mirror deflector 7a that changes the direction of the reflection mirror 7 may be employed as schematically illustrated in FIG. In the mirror deflector 7a, a motor 17 for rotating (spinning) the reflection mirror 7 is provided on the back surface of the reflection mirror 7. At this time, the mirror surface of the reflection mirror 7 is oriented in a direction inclined with respect to a plane perpendicular to the rotation axis thereof (that is, the rotation axis of the motor 17). The optical path passing through the inside is changed, and the position of the light detection region is moved. (When excitation light is irradiated, the traveling direction of the excitation light is changed with the rotation of the reflection mirror 7 and the condensing position is moved. Also, the light incident on the objective lens from the sample solution side is reflected. As the mirror 7 rotates, the conjugate position with the pinhole 13 moves, so that the region where light is detected, that is, the region in the object space imaged on the pinhole 13 moves. The movement path of the position of the light detection region obtained by rotating 7 is circular or elliptical. As will be described in detail later, in the present invention, in order to achieve a speed faster than the moving speed of the position of the light detection region achieved by the rotation of the reflection mirror 7 as described above, In addition, a mechanism for moving the position of the light detection region while oscillatingly displacing from the movement path achieved by the rotation of the reflection mirror 7 is further provided.

  Further, as an additional configuration, a stage (not shown) of the microscope is provided with a stage position changing device 17a for moving the horizontal position of the microplate 9 in order to change the well 10 to be observed. Good. The operation of the stage position changing device 17a may be controlled by the computer 18.

  When the luminescent particles emit light by multiphoton absorption, the above optical system is used as a multiphoton microscope. In that case, since there is light emission only in the focal region (light detection region) of the excitation light, the pinhole 13 may be removed. When the luminescent particles emit light by phosphorescence, the optical system of the confocal microscope is used as it is. Further, in the optical analyzer 1, as shown in the figure, a plurality of excitation light sources 2 may be provided, and the wavelength of the excitation light can be appropriately selected according to the wavelength of the light for exciting the luminescent particles. Good.

Improvement of mirror deflector As mentioned in the "Summary of the invention", when carrying out optical analysis technology such as scanning molecule counting method, the rotation speed of the mirror deflector motor is set to the maximum allowable speed. In some cases, it is desired to move the position of the light detection region at a speed faster than the movement speed of the position of the obtained light detection region. For example, in the scanning molecule counting method or FIDA, when the concentration of the luminescent particles to be observed in the sample solution is low, the scanning distance sufficient to obtain a good measurement result becomes long. If the moving speed of the mirror can be made faster than the moving speed of the position of the light detection region obtained by setting the rotation speed of the motor of the mirror deflector to an allowable maximum speed, it is advantageous that the measurement time is shortened. Therefore, in the present invention, as one embodiment, the structure of the reflection mirror 7 and the mirror deflector that rotates the reflection mirror 7 is improved.

  In the first embodiment of the improvement of the reflection mirror 7 and the mirror deflector, as schematically shown in FIG. 2A, the reflection mirror 7 is arranged on the annular member 20 on its inner periphery. A motor 17 that rotates the mirror 7 and the annular member 20 integrally is connected to the bottom of the annular member 20. Further, as schematically illustrated in FIGS. 2B and 2C, a tongue portion 32 along the longitudinal direction of the rod-like member extends downward on the back surface of the mirror base 24, and the tongue portion 32. On the other hand, the piezoelectric element 30 attached to the inner periphery of the annular member 20 is connected. The outer periphery of the annular member 20 is surrounded by a terminal electrode 34 that allows power to be supplied to the piezoelectric element 30 even while the annular member 20 is rotating. The terminal electrode 34 includes a power source (not shown). The terminal 34a that supplies power from

  In the operation of the mirror deflector carrying the reflecting mirror 7, the annular member 20 and the mirror 7 are integrally rotated by the rotation of the motor 17 (arrow a) as in the conventional apparatus. . Then, the light detection region moves along a circular or elliptical trajectory α indicated by a dotted line in FIG. However, in the illustrated embodiment, an oscillating voltage is further applied to the piezoelectric element 30 via the terminal electrode 34, whereby the piezoelectric element 30 is moved in the longitudinal direction (arrow c in FIG. 2C). The tongue 32 and the mirror pedestal 24 are swung by extending and contracting. When the tongue 32 and the mirror pedestal 24 swing, the rod-like member 22 that supports the mirror pedestal 24 vibrates and twists about its longitudinal axis. Then, since the mirror 7 rotates in the direction of the arrow a and further swings in the direction of the arrow b, the direction of the light reflected by the mirror 7 is also displaced, and FIG. As indicated by the solid line in D), the trajectory β is displaced from the trajectory α in the radial direction. Here, also when the light detection region follows the trajectory β, the movement period is the same as the period of one rotation of the motor 17, while the scanning distance of the trajectory β is obviously longer than the trajectory α. As described above, the mirror 7 oscillates in a vibrational manner, so that the moving speed of the position of the light detection region can be made faster than when the mirror 7 is simply rotated. Note that the voltage applied to the piezoelectric element and its frequency may be appropriately set experimentally or theoretically.

  FIG. 3 shows an embodiment of the configuration of another mirror deflector that realizes a trajectory similar to the trajectory β of FIG. First, in the example shown in FIG. 3A, the configurations of the annular member 20, the rod-like member 22 and the like are the same as in FIG. 2, but the piezoelectric element 30 is placed on the back surface of the mirror base 24. The rod-shaped member 22 is attached vertically to a portion shifted laterally with respect to the longitudinal axis of the rod-shaped member 22, and the piezoelectric element 30 is expanded and contracted in the direction of the arrow c while the motor 17 is rotating. Then, the rod-like member 22 that supports the mirror base 24 generates a torsional vibration about the longitudinal axis thereof, whereby the mirror 7 rotates in the direction of the arrow a and further in the direction of the arrow b. Since the vibration is oscillated, the direction of the light reflected by the mirror 7 is also displaced, and the light detection region is oscillating from the orbit α as shown by the solid line in FIG. The trajectory β displaced in the radial direction is followed.

  In the embodiment shown in FIG. 3B, a knurled convex portion or a sawtooth convex portion is provided over the entire outer periphery of the annular member 20, and the exterior of the annular member 20 is provided. Accordingly, the protruding member 42 formed of an arbitrary elastic material is brought into contact with the knurled groove (the piezoelectric element 30 and a configuration for driving the piezoelectric element 30 may not be provided). In the operation of such a configuration, when the motor 17 rotates, the tip of the projecting member 42 collides with a continuous knurled convex portion in order (arrow d), so that the annular member 20 is mechanically vibrated. Occur. Then, the mechanical vibration causes torsional vibration around the longitudinal axis of the rod-shaped member 22 in the rod-shaped member 22 and the mirror pedestal 24, and the mirror pedestal 24 carrying the mirror 7 vibrates in the direction of the arrow b. As a result, the direction of the light reflected by the mirror 7 is also displaced, so that the light detection region is vibrated from the trajectory α as shown by the solid line in FIG. The trajectory β displaced in the radial direction is followed. Note that the number and size of the knurled protrusions and the elasticity of the protruding member 42 may be selected experimentally or theoretically so as to obtain a desired vibrational displacement of the light detection region. When vibrational displacement of the light detection region is not necessary, the protruding member 42 is spaced from the knurled convex portion. Further, the knurled convex portion may be provided at any height position (intermediate, bottom, etc.) on the outer periphery of the annular member 20.

Increasing the moving speed of the position of the light detection region by oscillatingly displacing the position of the improved light detection region in the optical system from the trajectory α, that is, the path obtained when the mirror 7 is simply rotated. As another aspect, this can also be achieved by oscillatingly moving the position in the optical axis direction of the lens element, the pinhole, and the exit end of the optical fiber in the optical system back and forth. For example, as a first example, the lens element in the objective lens 8 is oscillatingly displaced back and forth. Specifically, as illustrated in FIG. 4A, a driving member 30a is attached to one of the lens elements 8a of the objective lens 8 from the outside of the lens barrel 8b. A piezoelectric element 30 arranged to expand and contract in the optical axis direction (arrow c) is bonded to the driving member 30a. As a result, when the piezoelectric element 30 expands and contracts, the lens element 8a is oscillatingly displaced back and forth. According to such a configuration, during the rotation of the motor 17, the light detection region is displaced from the orbital α in the optical axis direction when moving along the orbital α as depicted in FIG. will follow γ. Also in this case, as in the example of FIGS. 2 and 3, the moving period of the light detection region is the same as the period of one rotation of the motor 17, while the scanning distance of the orbit γ is clearly longer than the orbit α. The moving speed of the position of the light detection region is faster than when the mirror 7 is simply rotated.

  4E can also be achieved by oscillatingly displacing the positions of some optical elements in the optical axis direction at the site where the excitation light is introduced into the microscope optical system. is there. Specifically, the collimating lens 4 (FIG. 4B), the pinhole 4a for making the excitation light a point light source (FIG. 4C), or the optical fiber 3 for introducing the excitation light into the optical system. The tip 3a (FIG. 4D) is connected to the piezoelectric element 30 arranged so as to expand and contract in the optical axis direction (via the drive member 30a), and the piezoelectric element 30 is moved along with the rotation of the mirror 7 by the motor 17. When the telescopic movement is performed, the excitation light condensing region, that is, the light detection region moves so as to follow the trajectory γ of FIG. In this case, it is preferable that the position of the pinhole 13 on the light receiving side is also displaced in synchronization. Further, the improved configuration of the optical system in FIG. 4 may be combined with the improvements in the mirror deflector of FIG. In that case, the moving speed can be further increased.

  Thus, according to the present invention described above, when the mirror 7 is rotated by the motor 17, the position of the light detection region is substantially perpendicular to the traveling direction of the path formed by the rotation of the mirror 7 (path surface). (Vibration direction in the path or perpendicular to the optical axis), the scanning distance per cycle is increased, that is, the moving speed is increased, and a longer distance is scanned in a shorter time. Is achieved, and the time required for optical measurement is reduced.

Claims (6)

  An optical analyzer that detects and analyzes light from a light detection region in a sample using an optical system of a confocal microscope or a multiphoton microscope, and the direction of the optical path that enters and exits from the back of the objective lens by rotating. The position of the light detection region is vibrated from the optical path deflection optical element that moves the position of the light detection region by changing the position of the light detection region formed by rotation of the optical path deflection optical element. And a light detection area moving path displacing means for displacing the light detection area.   2. The apparatus according to claim 1, wherein the optical element for deflecting an optical path has a rotation axis and a mirror surface extending in a direction inclined with respect to a plane perpendicular to the rotation axis, and is driven to rotate around the rotation axis. The light detection area moving path displacing means oscillates the direction of the mirror surface during rotation of the deflection mirror, and the position of the light detection area oscillates from the path. A device characterized by.   3. The apparatus according to claim 2, wherein the light detection area moving path displacement means extends in a plane perpendicular to the rotation axis along the outer periphery of the deflection mirror and rotates integrally with the deflection mirror. And a rod-shaped member extending in the diameter direction from the inner periphery of the annular member and connected to the deflection mirror, and means for generating torsional vibration in the rod-shaped member with the extending direction of the rod-shaped member as an axis, The apparatus according to claim 1, wherein the mirror surface of the deflection mirror is rotated about the axis in the extending direction of the rod member by torsional vibration of the rod member during rotation of the deflection mirror.   4. The piezoelectric element according to claim 3, wherein the means for generating torsional vibration in the rod-shaped member applies a vibrational rotational force around an axis in the extending direction of the rod-shaped member by contacting the back surface of the deflection mirror. A device characterized by being.   4. The apparatus according to claim 3, wherein a knurled groove formed on an outer periphery or a back surface of the annular member is formed, and means for generating a twisting motion on the rod member is rotated integrally with the annular member. A projecting member that comes into contact with the knurled groove and mechanically vibrates when the projecting member collides with the knurled continuous convex portion in turn during the rotation of the deflecting mirror and the annular member. Thereby generating torsional vibrations of the rod-shaped member.   2. The apparatus according to claim 1, wherein the light detection region moving path displacing means has at least one position of a lens in the optical system or a tip of an optical fiber emitting excitation light or a pinhole in an optical axis direction. An apparatus for oscillating and displacing the position of the light detection region from the path in the direction of the optical axis.

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