WO2017221356A1 - Microscope - Google Patents

Microscope Download PDF

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Publication number
WO2017221356A1
WO2017221356A1 PCT/JP2016/068560 JP2016068560W WO2017221356A1 WO 2017221356 A1 WO2017221356 A1 WO 2017221356A1 JP 2016068560 W JP2016068560 W JP 2016068560W WO 2017221356 A1 WO2017221356 A1 WO 2017221356A1
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WO
WIPO (PCT)
Prior art keywords
fluorescence
positional relationship
light
excitation light
sample
Prior art date
Application number
PCT/JP2016/068560
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English (en)
Japanese (ja)
Inventor
兼太郎 井元
厚志 土井
Original Assignee
オリンパス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by オリンパス株式会社 filed Critical オリンパス株式会社
Priority to PCT/JP2016/068560 priority Critical patent/WO2017221356A1/fr
Priority to JP2018523216A priority patent/JPWO2017221356A1/ja
Publication of WO2017221356A1 publication Critical patent/WO2017221356A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

Definitions

  • the present invention relates to a microscope.
  • the positional relationship between the confocal pinhole and the focal point of the excitation light is optically determined.
  • a microscope is known that detects fluorescence by switching between a conjugate position and a non-conjugated position, and calculates the difference between the obtained fluorescence signals (see, for example, Patent Document 1).
  • a scanner that scans excitation light from a light source, and the excitation light that is scanned by the scanner is collected on a sample, while signal light generated in the sample is collected at each scanning position.
  • An objective optical system, a detector for detecting the signal light collected by the objective optical system, and the signal light arranged between the detector and the objective optical system and condensed by the objective optical system And a positional relationship between the light shielding member and the condensing point of the excitation light in the sample in terms of an optically conjugate positional relationship and an optically non-conjugated positional relationship.
  • a switching unit that automatically switches, a fluorescence distribution acquisition unit that acquires a fluorescence distribution at a position optically conjugate with the condensing point, and the switching unit based on the fluorescence distribution acquired by the fluorescence distribution acquisition unit.
  • a fluorescence distribution acquisition unit that acquires a fluorescence distribution at a position optically conjugate with the condensing point
  • the switching unit based on the fluorescence distribution acquired by the fluorescence distribution acquisition unit.
  • the excitation light from the light source is scanned by the scanner and condensed on the sample by the objective optical system, whereby the fluorescent material is excited at the collection point of the excitation light in the sample to generate fluorescence.
  • the generated fluorescence and the reflected light of the excitation light from the sample are collected by the objective optical system and then detected by the detector, and a fluorescence image is generated by associating the detected fluorescence intensity with the scanning position.
  • the fluorescence distribution acquisition unit acquires a fluorescence distribution at a position optically conjugate with the condensing point, and the setting unit sets a non-conjugated positional relationship in the switching unit based on the acquired fluorescence distribution.
  • the positional relationship between the light blocking member provided in the previous stage of the detector and the condensing point of the excitation light in the sample is switched to a conjugate positional relationship by the operation of the switching unit, the focus fluorescence and the out-of-focus fluorescence are detected. Detected by the instrument.
  • the focused fluorescence cannot pass through the light shielding member, and only the out-of-focus fluorescence is detected by the detector. Then, by calculating the difference between these detected fluorescence signals, it is possible to obtain the focused fluorescence from which the out-of-focus fluorescence has been removed, and to obtain a clear fluorescence image.
  • the operator since an optimal non-conjugated positional relationship can be obtained based on the fluorescence distribution at a position optically conjugate with the condensing point, the operator can perform focusing without performing complicated setting work. It is possible to obtain focused fluorescence from which external fluorescence has been accurately removed. Therefore, the burden on the operator can be reduced.
  • the setting unit separates the fluorescence distribution acquired by the fluorescence distribution acquisition unit into a focus fluorescence and an out-of-focus fluorescence, and the focus fluorescence falls below a predetermined threshold and
  • the positional relationship closest to the optically conjugate position may be set as the non-conjugated positional relationship in the switching unit.
  • the said setting part may apply the said fluorescence distribution acquired by the said fluorescence distribution acquisition part to a predetermined distribution model, and may isolate
  • the said fluorescence distribution acquisition part changes the positional relationship of the said condensing point of the said excitation light in the said sample, and the said light-shielding member
  • the said signal light detected by the said detector The fluorescence distribution may be acquired based on the fluorescence intensity.
  • the fluorescence distribution acquisition unit is configured such that the positional relationship between the light shielding member and the condensing point of the excitation light in the sample is a conjugate positional relationship, and two different non-conjugated conjugates.
  • fluorescence intensity is acquired at least at three points: a conjugate positional relationship, a positional relationship with a high degree of non-conjugation, and a positional relationship with a low degree of non-conjugation that is close to the conjugate positional relationship.
  • a conjugate positional relationship a positional relationship with a high degree of non-conjugation
  • a positional relationship with a low degree of non-conjugation that is close to the conjugate positional relationship.
  • the fluorescence distribution acquisition unit is based on a theoretical size of the spot diameter of the excitation light at the condensing point determined by the objective optical system and the wavelength of the excitation light. You may set the positional relationship of the said light shielding member which acquires the intensity
  • positioned in the position optically conjugate with the said condensing point of the said excitation light in the said sample may be sufficient as the said fluorescence distribution acquisition part.
  • the fluorescence distribution acquisition unit acquires the fluorescence distribution at a plurality of points
  • the setting unit determines the non-conjugated positional relationship in the switching unit based on an average of the plurality of fluorescence distributions. It may be set. In this way, it is possible to easily set a non-conjugated positional relationship in which the focused fluorescence and the out-of-focus fluorescence can be accurately separated by the setting unit without acquiring a fluorescence image, and excitation to the sample. It is possible to reduce the damage of the sample by reducing the light irradiation time.
  • FIG. 2 is a diagram illustrating an example of an irradiation pattern of one of the two types of excitation light set by the setting unit in the imaging mode of the microscope of FIG. 1.
  • FIG. 3 is a diagram illustrating an example of an irradiation pattern of the other of the two types of excitation light set by the setting unit in the imaging mode of the microscope of FIG. It is a block diagram which shows the 1st modification of the microscope of FIG. It is a block diagram which shows the 2nd modification of the microscope of FIG. It is a top view which shows the disk which has the pinhole of the microscope of FIG. 6A. It is a block diagram which shows the 3rd modification of the microscope of FIG. It is a block diagram which shows the 4th modification of the microscope of FIG. It is a block diagram which shows the 5th modification of the microscope of FIG.
  • a microscope 1 according to an embodiment of the present invention will be described below with reference to the drawings.
  • the microscope 1 according to the present embodiment is switched by the switching unit 3 that switches the excitation light from the laser light source 2 to two types of excitation light that are alternately emitted, and the switching unit 3.
  • a microscope body 4 that irradiates the sample A with two types of excitation light and detects the fluorescence generated in the sample A, and a calculation unit 5 that generates an image by calculation using the fluorescence detected in the microscope body 4;
  • a monitor 6 for displaying an image generated by the calculation unit 5.
  • the microscope body 4 condenses the excitation light scanned by the scanner 7 in a two-dimensional manner and the excitation light scanned by the scanner 7 on the sample A, and the fluorescence (signal light) from the sample A.
  • a converging objective lens (objective optical system) 8 a dichroic mirror 9 for diverging fluorescence collected by the objective lens 8 and returning via the scanner 7 from the optical path of the excitation light, and branched by the dichroic mirror 9
  • An imaging lens 10 that condenses the fluorescent light, a pinhole (light-shielding member) 11 that is optically conjugate with the focal position of the objective lens 8, and light that detects the fluorescent light that has passed through the pinhole 11
  • pinhole 11 was illustrated as a light shielding member, it replaces with this, and when it arrange
  • Any light shielding member that blocks the focal fluorescence when the light is emitted may be used.
  • examples of the light shielding member include a micromirror device and a spatial light modulator.
  • the scanner 7 is, for example, a proximity galvanometer mirror in which two galvanometer mirrors that can be swung around a non-parallel axis line are arranged close to each other.
  • the photodetector 12 is, for example, a photomultiplier tube (PMT).
  • the laser light source 2 is a light source that continuously emits excitation light.
  • the switching unit 3 includes a movable mirror (deflection element) 13 that changes the swing angle, and a drive control unit 14 that drives the movable mirror 13.
  • a setting unit 16 is connected to the drive control unit 14, and an input unit 17 and a distribution acquisition unit (fluorescence distribution acquisition unit) 18 are connected to the setting unit 16.
  • the input unit 17 is configured by a keyboard, a mouse, or a GUI that performs input for selecting two operation modes of a setting mode and a shooting mode. Based on the input from the input unit 17, the setting unit 16 sets the drive control unit 14 to operate in one of two operation modes.
  • the setting unit 16 When the setting mode is selected from the input unit 17, the setting unit 16 operates the drive control unit 14 so as to drive the movable mirror 13 and shift the laser beam by a preset shift amount.
  • the drive control unit 14 When the photographing mode is selected, the drive control unit 14 is caused to function as a frequency oscillator that oscillates a predetermined frequency.
  • the preset shift amount x is the first shift amount with zero shift amount in which the condensing point and the pinhole 11 are in an optically conjugate positional relationship.
  • x1 the second shift amount x2 where the condensing point and the pinhole 11 are partially optically non-conjugated, and the positional relationship where the condensing point and the pinhole 11 are sufficiently optically non-conjugated.
  • the third shift amount x3 The third shift amount x3.
  • the setting unit 16 instructs the drive control unit 14 to set the movable mirror 13 at an angle at which three preset shift amounts can be achieved, and the movable mirror 13.
  • the intensity of the fluorescence detected by the photodetector 12 at each angle is stored in the distribution acquisition unit 18.
  • the distribution acquisition unit 18 generates a fluorescence distribution in which the shift amount x that can achieve each of the three angles of the movable mirror 13 is associated with the fluorescence intensity acquired at each angle.
  • the fluorescence detected by the photodetector 12 is shown in FIG.
  • the out-of-focus fluorescence generated at a position other than the focus is also included, while the focused fluorescence generated from the focus is largely dominant.
  • the focal fluorescence decreases, but the out-of-focus fluorescence does not change much.
  • the setting unit 16 can achieve the positional relationship between the condensing point and the pinhole 11 that can detect the fluorescence in which the out-of-focus fluorescence is dominant and hardly includes the focused fluorescence from the fluorescence distribution acquired by the distribution acquisition unit 18.
  • angle of a movable mirror 13 that is, in order to calculate the achievable angle of the shift amount of the code x P in Figure 2B.
  • the method of calculating the shift amount of achievable angle of the code x P is the fluorescence distribution of three points generated by the distribution obtaining unit 18, for example, how to find an optimum position by fitting the distribution model such as a Gaussian function Can be mentioned.
  • the focal fluorescence of the first term and the out-of-focus fluorescence of the second term can be separated.
  • P can be set as an optimal non-conjugated positional relationship by the switching unit 3. Although 0.01 means that the focal fluorescence has decreased to 1%, this value can be set arbitrarily.
  • the angle of the movable mirror 13 to be set is set in the drive control unit 14 as the angle of the movable mirror 13 in the photographing mode.
  • the drive control unit 14 oscillates a predetermined frequency, and in synchronization with the oscillated frequency, the angle of the movable mirror 13 is alternately switched between the two angles set by the setting unit 16. It has become.
  • Reference numeral 15 in the figure denotes a mirror.
  • the movable mirror 13 was illustrated as a deflection
  • the two types of excitation light incident on the microscope body 4 are formed in a rectangular wave shape having inverted timings as shown in FIGS. 4A and 4B.
  • the rectangular wave-like light which has the timing reversed as two types of excitation light was illustrated here, instead of this, it has arbitrary repetitive shapes, such as a sine wave shape, and employ
  • One excitation light shown in FIG. 4A is focused on a position optically conjugate with the pinhole 11 via the scanner 7 and the objective lens 8, and the other excitation light shown in FIG.
  • the focal point is focused on a position optically unconjugated with the pinhole 11 via the lens 8.
  • the incident angle switching frequency is set to a frequency at which two types of excitation light can be blinked at least once at each pixel position.
  • the calculation unit 5 calculates the difference in the intensity of the fluorescence generated by the two types of excitation light detected by the photodetector 12 at the same pixel position.
  • the computing unit 5 includes, for example, a lock-in amplifier (not shown).
  • the lock-in amplifier calculates the difference between the two types of fluorescence signals output from the photodetector 12 in hardware in synchronization with the frequency generated by the drive control unit 14 for blinking the excitation light. ing. Then, the calculation unit 5 generates an image by storing the difference calculated for each pixel and the scanning position by the scanner 7 in association with each other.
  • the sample A is arranged on the stage (not shown) of the microscope body 4 so that the focal position of the objective lens 8 coincides with the sample A. Then, continuous excitation light is generated from the laser light source 2.
  • the setting unit 16 When the setting mode is selected in the input unit 17, the setting unit 16 is different from the drive control unit 14 in the shift amount x1 in which the condensing point and the pinhole 11 are conjugate and the degree of nonconjugation is different.
  • the angle of the movable mirror 13 is set to three angles corresponding to the two shift amounts x2 and x3, and the fluorescence intensity detected by the photodetector 12 at each angle is associated with the shift amount x by the distribution acquisition unit 18. And a fluorescence distribution is generated.
  • the generated fluorescence distribution is sent to the setting unit 16.
  • the angle of the movable mirror 13 is calculated.
  • the setting unit 16 sets the calculated angle of the movable mirror 13 as the angle of the movable mirror 13 that is switched by the drive control unit 14 when the photographing mode is selected by the input unit 17.
  • the drive control unit 14 alternately changes the angle of the movable mirror 13 to two angles set by the setting unit 16 according to a predetermined frequency oscillated by the drive control unit 14. Two types of excitation light having different incident angles are alternately incident on the main body 4.
  • the focal fluorescence contained in one excitation light is condensed at a position in the sample A conjugate with the pinhole 11, so that the fluorescence generated in the vicinity of the focal position is condensed by the objective lens 8, On the way back through the scanner 7, it is separated by the dichroic mirror 9, condensed by the imaging lens 10, passes through the pinhole 11, and is detected by the photodetector 12.
  • the excitation light excites the fluorescent substance by passing through the sample A even in the middle of the path to the focal position of the objective lens 8. This occurs not only in the focal position of the lens but also in the middle of the path to the focal position.
  • the sample A is made of a scattering material, the fluorescence tends to be generated at a site other than the focal position due to scattering of the excitation light.
  • the fluorescence generated from the focal position of the objective lens 8 easily passes through the pinhole 11 disposed at the optically conjugate position, and thus is detected as signal light by the photodetector 12.
  • the fluorescence generated from the part other than the focal position is scattered by the sample, and a part thereof passes through the pinhole 11 and is detected as noise by the photodetector 12. Therefore, the fluorescence detected by the irradiation of one excitation light includes the focal fluorescence generated at the focal position of the objective lens 8 and to be acquired as a signal, and the out-of-focus that is generated at the other part and should not be acquired as a signal. Fluorescence and are included.
  • the other excitation light is condensed at a position unconjugated with the pinhole 11 in the sample A, and excites the fluorescent substance in the middle of the path to the focal position and the focal position of the objective lens 8. This generates fluorescence.
  • the fluorescence generated at the non-conjugated focal position cannot be passed through the pinhole 11 and is blocked, while a part of the fluorescence generated from the part other than the focal position is scattered by the sample.
  • the light passes through the same pinhole 11 and is detected by the photodetector 12.
  • the focal fluorescence in the fluorescence detected by the photodetector 12 is reduced by 99% and the focal point is reduced.
  • Outer fluorescence is substantially equivalent to out-of-focus fluorescence detected by the photodetector 12 in a conjugate positional relationship. Therefore, the calculation unit 5 calculates the difference between the fluorescence detected by the irradiation of these two types of excitation light, thereby generating a focal point that should not be acquired as a signal, generated in a part other than the focal position of the objective lens 8.
  • the focal fluorescence from which the external fluorescence is removed can be obtained with high accuracy.
  • the range in which fluorescence is generated by irradiating two types of excitation light is not exactly the same, but it is the same in many parts, and detection is performed using the same pinhole 11 Thus, most of the out-of-focus fluorescence can be removed by subtracting as it is. In particular, when high-definition observation is performed by increasing the NA of excitation light, the overlap ratio of the fluorescence generation range increases, so that the out-of-focus fluorescence can be more effectively removed.
  • the fluorescence generated at the focal position of the objective lens 8 can be detected with a high S / N ratio, and a clear image with little noise can be acquired.
  • the effect is high at the time of high-definition observation with a large NA of excitation light and when the sample A is a strong scattering material and easily generates out-of-focus fluorescence. Since the optimum non-conjugate position is automatically set, the operator does not need to perform the troublesome work of manually adjusting the positional relationship between the pinhole 11 and the fluorescent light beam while photographing the sample A. There is an advantage that the burden can be reduced.
  • the pinhole 11 is exemplified as the light shielding member, but instead of this, when the focus hole is arranged at a position optically conjugate with the focus position of the objective lens 8, the focused fluorescence is passed,
  • An arbitrary light shielding member that blocks the focal fluorescence when arranged at a conjugate position may be employed.
  • the other light shielding member include a micromirror device and a spatial light modulator.
  • the signal light from the sample A detected by the photodetector 12 is exemplified using only fluorescence, but in addition to this, reflected light of excitation light from the sample A is used. May be.
  • the refractive index distribution can be imaged.
  • the movable mirror 13 is exemplified as the deflecting element constituting the switching unit 3, as shown in FIG. 5, the acousto-optic deflector (acousto-optic element, beam moving unit) 19 or the electro-optic deflector (electro-optic element, beam).
  • a device such as (moving unit) 20 can also be used.
  • These devices 19 and 20 also switch the voltage input according to a predetermined frequency oscillated by the drive control unit 14, so that the fluorescent light beam enters the imaging lens 10 in synchronization with the input voltage in the same manner as the movable mirror 13. The angle can be changed.
  • the setting unit 16 switches the voltage generated by the drive control unit 14 during the setting mode, causes the distribution acquisition unit 18 to acquire the fluorescence distribution, and inputs the fluorescence distribution to the devices 19 and 20 during the imaging mode based on the acquired fluorescence distribution. Set the voltage. Since these devices 19 and 20 do not include a movable part such as the movable mirror 13, the device can be configured to be compact and have a long life.
  • the incident position of the fluorescent light beam incident on the fixed light shielding member 11 is switched over time.
  • the fluorescent light beam is fixed and the light shielding member 21 is fixed. May be moved in a direction crossing the optical axis S of the fluorescent light beam. That is, as shown in FIG. 6B, a disk-shaped disk having a plurality of pinholes 22 arranged at intervals in the circumferential direction is adopted as the light shielding member 21, and a motor is used as shown in FIG. 6A.
  • the (switching unit) 23 may rotate the disk 21 around the central axis.
  • the fluorescence condensing position by the imaging lens 10 is made coincident with the disk 21, and the disk 21 is rotated by the motor 23 so that the pinhole 22 coincides with the optical axis S of the fluorescence.
  • a state that does not match can be alternately repeated. That is, in a state where any one of the pinholes 22 coincides with the fluorescence optical axis S, the focal point of the excitation light in the sample A and the pinhole 22 are in an optically conjugate positional relationship.
  • the condensing point of the excitation light in the sample A and the pinhole 22 are in an optically non-conjugated positional relationship.
  • the fluorescence distribution is calculated based on the fluorescence intensities acquired at the three positions of the position where the pinhole 22 is aligned with the optical axis S of the fluorescence, the position where the pinhole 22 is partially matched, and the position which is not completely matched.
  • an optically conjugate positional relationship and an optically non-conjugated positional relationship are alternately formed in time, and two types of fluorescence for accurately detecting the focused fluorescence are detected by the same photodetector 12. Can be detected sequentially. Further, there is an advantage that the two positional relationships can be switched at a higher speed by rotating the disk 21 at a higher speed.
  • the case where the fluorescent light beam is moved in the direction intersecting the optical axis S with respect to the pinhole 22 is exemplified, but instead, the pinhole 22 is moved with respect to the fluorescent light beam. It may be.
  • the non-conjugated positional relationship may be achieved by making the incident angle of the excitation light incident on the sample A from the laser light source 2 different. In that case, when the incident angle of the excitation light is changed by the movable mirror 30 arranged between the laser light source 2 and the dichroic mirror 9, the scanner 7 is moved so that the position of the condensing point in the sample A does not shift. It is necessary to operate in conjunction.
  • Reference numeral 31 denotes a mirror for forming an optical path of excitation light from the laser light source 2 to the movable mirror 30.
  • the distribution model for fitting is not limited to a Gaussian function, and a Lorentz function or a Forked function may be used.
  • a fluorescence distribution may be generated by detecting the fluorescence intensity in the quantity. Thereby, the precision of fitting to a distribution model can be improved.
  • the fluorescence distribution may be acquired at one location in the sample A, or the fluorescence distribution may be generated by averaging the fluorescence intensities acquired at two or more locations.
  • x x2 and x3 are set in advance, but as a guide, Airy (AIRY) which is a unit representing the theoretical size of the spot diameter of the excitation light may be used.
  • AIRY Airy
  • the shift amount x2 0.5 air
  • the shift amount x3 5 air You can set it to. In this way, it is possible to calculate the shift amount that optimizes the non-conjugated positional relationship with high accuracy by the three fluorescence distributions.
  • the fluorescence intensity detected using the photodetector 12 provided in the microscope body 4 is also used when setting the shift amount in the setting mode. Thereby, the fluorescence distribution can be generated without adding a new device to the basic configuration of the microscope body 4.
  • a half mirror 24 that branches a part of the fluorescence from the sample A, and a camera that images the branched fluorescence at a position optically conjugate with the pinhole 11 A two-dimensional sensor such as a fluorescence distribution acquisition unit 25 may be provided.
  • reference numeral 26 denotes a condenser lens.
  • the fluorescent light beam and the pinhole 11 are relatively moved in the direction intersecting the optical axis S of the fluorescent light beam. Instead, as shown in FIG.
  • the fluorescent light beam and the pinhole 11 may be relatively moved in the direction along the optical axis S of the fluorescent light beam.
  • an acousto-optic lens 27 is employed instead of the acousto-optic deflector 19 or the electro-optic deflector 20.
  • the acousto-optic lens 27 is a lens that changes the refractive power in accordance with the input voltage.
  • the imaging position of the fluorescence by the imaging lens 10 is changed.
  • the position can be switched between a position matching the pinhole 11 and a position shifted from the pinhole 11 in the optical axis direction. Even in this case, the optimum non-conjugated positional relationship exists, and the out-of-focus fluorescence can be accurately removed by setting the optimal positional relationship.

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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
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  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Selon la présente invention, dans le but de régler automatiquement un point optimal qui permet à un trou d'épingle confocal et à un point de collecte de la lumière d'excitation d'être dans une relation de position optiquement non conjuguée, et de soulager une charge sur un opérateur, un microscope (1) comprend : un scanner (7) qui irradie une lumière d'excitation destinée au balayage à partir d'une source de lumière (2); un système optique d'objectif (8) qui collecte, au niveau d'un échantillon (A), une lumière d'excitation destinée au balayage et collecte, dans chaque position de balayage, une lumière fluorescente générée au niveau de l'échantillon; un détecteur (12) qui détecte la lumière fluorescente collectée; un élément de protection contre la lumière (11) qui est disposé entre le détecteur et le système optique d'objectif et qui protège partiellement la lumière fluorescente collectée par le système optique d'objectif; une unité de commutation (3) qui commute temporellement, entre une relation de position optiquement conjuguée et une relation de position optiquement non conjuguée, une relation de position entre l'élément de protection contre la lumière et le point de collecte de la lumière d'excitation au niveau de l'échantillon; une unité d'acquisition de la distribution de lumière fluorescente (18) qui acquiert une distribution de lumière fluorescente dans une position optiquement conjuguée par rapport au point de collecte de lumière; et une unité de réglage (16) qui établit une relation de position non conjuguée dans l'unité de commutation sur la base de la distribution de lumière fluorescente acquise.
PCT/JP2016/068560 2016-06-22 2016-06-22 Microscope WO2017221356A1 (fr)

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PCT/JP2016/068560 WO2017221356A1 (fr) 2016-06-22 2016-06-22 Microscope
JP2018523216A JPWO2017221356A1 (ja) 2016-06-22 2016-06-22 顕微鏡

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009510498A (ja) * 2005-09-29 2009-03-12 カール ツァイス マイクロイメージング ゲーエムベーハー 顕微鏡検査方法および顕微鏡
WO2015163261A1 (fr) * 2014-04-24 2015-10-29 オリンパス株式会社 Microscope et procédé d'observation microscopique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009510498A (ja) * 2005-09-29 2009-03-12 カール ツァイス マイクロイメージング ゲーエムベーハー 顕微鏡検査方法および顕微鏡
WO2015163261A1 (fr) * 2014-04-24 2015-10-29 オリンパス株式会社 Microscope et procédé d'observation microscopique

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