JP2010145950A - Liquid-immersion objective lens and microscope including the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid-immersion objective lens capable of enhancing performance of optical tweezers, and to provide a microscope including the liquid-immersion objective lens. <P>SOLUTION: In the liquid-immersion objective lens 6 whose NA is higher than the refractive index of water, the lens 6 includes an optical member having a dichroic film 7 which acts only for a light beam having NA ≥naO (where a condition of na0≥1.3 is satisfied), and transmits only a light beam having a wavelength of ≤λ0 (where a condition of λ0≤800 nm is satisfied). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an immersion objective lens suitable for optical tweezers and a microscope having the immersion objective lens.

  A fine particle manipulation method using light that captures and manipulates fine particles in a medium by light irradiation is generally referred to as optical tweezers or an optical trap. Since a laser is mainly used as a light source, it is also referred to as a laser tweezer or laser trapping. In optical tweezers, laser light (trap light) from a light source is condensed by a condensing optical system, for example, an objective lens of a microscope, and the condensed light beam is irradiated onto the fine particles in the medium, thereby generating light generated in the fine particles. Captures, holds and moves fine particles using radiation pressure. Optical tweezers are used in various fields as a method for capturing and manipulating living cells and microorganisms in a non-contact and non-destructive manner (see, for example, Patent Document 1).

  In recent years, optical tweezers have been widely used in the field of biological research using a microscope. Specifically, a microparticle is attached to a specific part of a cell, for example, a spring structure, and the microparticle is captured and pulled by optical tweezers. The spring constant is measured to measure the force generated in the cell, or the cell is identified. It is performed to move particles or to select specific cells from a plurality of cells.

  In such a biological research field, in order to prevent the trap light of the optical tweezers collected by the objective lens from damaging the cell, infrared light that is hardly absorbed by the cell is often used as the trap light. A high-power YAG laser (wavelength 1064 nm) or the like is used. In addition, observation of captured microparticles and biological samples is not limited to bright-field observation with transmitted illumination, but is often performed with fluorescence in order to observe captured microparticles, which are often very small, with good contrast. The wavelengths of illumination light (transmission illumination light and excitation light for performing fluorescence observation) and observation light (transmission light and fluorescence) are often in the visible range.

  In such optical tweezers in the field of biological research, generally, the higher the numerical aperture (hereinafter referred to as NA) of an objective lens that collects trapped light, the greater the capturing power for capturing fine particles. In addition, since the captured fine particles are often fluorescent fine particles in water, incidental fluorescence observation is often used in addition to bright field observation as a means of observing the captured fine particles, and an observer side who obtains as bright a fluorescent image as possible. Therefore, it is desirable that the objective lens has a high NA. As a result, an immersion objective lens such as a water immersion objective lens or an oil immersion objective lens is often used for the optical tweezers.

Furthermore, if the fluorescent fine particles are small, or if you want to observe the fine particles near the interface between the sample cover glass and water (culture solution) with a good S / N, capture using total reflection fluorescence observation (TIRF) Often, observed fine particles are observed. In that case, the excitation light, which is the illumination light for observation, is often used with an objective illumination system that enters the sample through the objective lens from the viewpoint of convenience, but is incident at an angle that causes total reflection at the interface between the sample cover glass and water. Therefore, the NA of the objective lens to be used needs to be at least higher than 1.33 (the refractive index of water). In practice, an oil immersion objective lens having an NA of 1.4 or more is often used.
JP 2002-303800 A

  When an immersion objective lens with an NA higher than the refractive index of water is used, the light of which the NA is higher than the refractive index of water out of the trap light causes total reflection at the interface between the sample cover glass and the water, and the vicinity of the interface. The evanescent field is a permanent place. In general, trap light is often used with higher power than excitation light for fluorescence observation, so that an evanescent field much stronger than an evanescent field generated only by ordinary fluorescence observation or TIRF is generated at the interface.

  This evanescent field generates a pulling force on the fine particles when the fine particles captured by the trapped light are present near the interface. As a result, an external force other than the original trapping force acts on the fine particles, which has a problem of adversely affecting the measurement of the spring constant and the manipulation of the fine particles.

  In order to avoid such a problem, there is a method in which an aperture is provided in the optical path for introducing trap light to limit the NA of the trap light incident on the objective lens. However, this method has a problem that it is necessary to provide a plane conjugate with the rear focal plane of the objective lens in the optical path for introducing trapped light and to set a stop there, resulting in a large and complicated apparatus. In addition, in order to perform stable trapping, it is necessary that the intensity distribution of the trapped light incident on the objective lens has good symmetry with respect to the optical axis center of the objective lens, and the pupil of the objective lens via many relay systems. There is a problem that fine adjustment such as centering of the stop is indispensable when focusing the light beam on a conjugate plane away from the center.

  The present invention has been devised to solve the above-mentioned problems, and aims to provide an immersion objective lens capable of enhancing the performance of optical tweezers, and further provides a microscope having this immersion objective lens. The purpose is to do.

In order to solve the above-described problems, the immersion objective lens according to the present invention acts only on a NA light beam of NA0 or more that satisfies the following conditions in an immersion objective lens whose NA is higher than the refractive index of water. And an optical member provided with a dichroic film that transmits only light having a wavelength of λ0 or less that satisfies the above condition.
na0 ≧ 1.3
λ0 ≦ 800nm

  In addition, a microscope according to the present invention includes the immersion objective lens.

  According to the present invention, the performance of optical tweezers can be improved.

  Hereinafter, an immersion objective lens according to an embodiment of the present application and a microscope having the immersion objective lens will be described with reference to the drawings.

  FIG. 1 is a diagram schematically showing a configuration of a microscope having an immersion objective lens according to the present embodiment.

  FIG. 2 is a diagram for explaining the principle of optical tweezers using the immersion objective lens of the present embodiment.

  FIG. 3 is a view showing an optical member provided with a dichroic film provided in the immersion objective lens of the present embodiment.

  First, the configuration of a microscope having the immersion objective lens 6 of this embodiment will be described.

  As shown in FIG. 1, the microscope having the immersion objective lens 6 of the present embodiment is a transmission optical microscope provided with an apparatus capable of fine particle manipulation by optical tweezers. The form of the microscope is not particularly limited, and may be an epi-illumination fluorescent microscope, a total reflection illumination fluorescent microscope, or the like. The microscope having the immersion objective lens 6 of the present embodiment includes a light source 1 for supplying trap light. The light source 1 is, for example, a high-power YAG laser and supplies infrared laser light having a wavelength of 1064 nm.

  The light supplied from the light source 1 enters a light guide 2 such as an optical fiber, and propagates through the light guide 2 and then exits from the exit end. The divergent light from the exit end of the light guide 2 becomes a substantially parallel light beam through the collector lens 3 and enters the dichroic mirror 4. Here, the collector lens 3 is configured to be movable along the optical axis AX by the action of the drive unit 5. Further, the dichroic mirror 4 has a characteristic of reflecting light from the light source 1 in a wavelength selective manner.

  The trapped light from the light source 1 is reflected by the dichroic mirror 4 and then travels toward the immersion objective lens 6 having a NA higher than the refractive index of water 1.33 and having a well corrected spherical aberration. Here, the immersion objective lens 6 of the present embodiment is provided with a dichroic film 7 in the vicinity of the rear focal plane of the immersion objective lens 6. The dichroic film 7 is deposited on the plane parallel plate 8 as shown in FIG. 2, but the plane parallel plate 8 is not shown in FIG. The characteristics of the dichroic film 7 and the action on the trap light will be described in detail later. However, the dichroic film 7 restricts the luminous flux of the trap light and restricts the NA.

  The trapped light is condensed near the front focal position by the immersion objective lens 6 and applied to the fine particles S in the medium B positioned in the vicinity of the focal position. The medium B containing the fine particles S is water (culture solution) held by a holder (not shown) such as a petri dish or a slide glass and a cover glass (not shown) that covers the upper part thereof. Note that the space between the immersion objective lens 6 and the cover glass is filled with immersion liquid (not shown) such as immersion oil (refractive index 1.51).

  In addition, the microscope having the immersion objective lens 6 according to this embodiment includes a transmitted illumination light source 11 for observing captured fine particles and biological samples. The transmitted illumination light source 11 is a halogen lamp, for example, and supplies visible light. The illumination light from the transmitted illumination light source 11 illuminates the medium B and the fine particles S therein through the illumination optical system 12, as indicated by a dotted line in FIG. The observation light (visible light transmitted through the medium B) from the illuminated fine particles S is a light beam having a diameter larger than that of the trapped light between the fine particles S and the dichroic film 7, as shown by a solid line in FIG. It becomes a substantially parallel light beam through the lens 6, passes through the dichroic film 7 (the action of the dichroic film 7 on the observation light will be described in detail later), and enters the dichroic mirror 4. Here, the dichroic mirror 4 has a characteristic of selectively transmitting light from the transmissive illumination light source 11 with a wavelength.

  The observation light from the fine particles S illuminated by the illumination light from the transmitted illumination light source 11 passes through the dichroic mirror 4 and then forms an image of the fine particles S on the predetermined image plane 14 via the second objective lens 13. The image of the fine particles S formed on the image plane 14 is observed by the naked eye 17 via an imaging means 15 such as a CCD camera positioned with respect to the image plane 14 and an eyepiece 16. In this way, the observer uses the transmitted illumination light source 11, the illumination optical system 12, the immersion objective lens 6, the second objective lens 13, the imaging means 15, and / or the eyepiece lens 16 that constitute the observation system, so that the observer can remove the fine particles S. Can be observed. The same applies to the observation of the biological sample in the medium B other than the captured fine particles S.

  Next, the principle of optical tweezers using the immersion objective lens 6 of this embodiment will be described.

  FIG. 2 is an enlarged view of the immersion objective lens 6 and the fine particles S of FIG. In FIG. 2, not only the immersion liquid and the cover glass but also the medium B is omitted. As shown in FIG. 2, the substantially parallel light beam L <b> 1 of the trap light is focused by the dichroic film 7 deposited on the upper surface of the plane parallel plate 8, and then enters the immersion objective lens 6. Thus, the light is condensed at a point P on the optical axis AX. In this way, the condensed light beam (for example, the conical or conical condensed light beam) L11 generated through the immersion objective lens 6 is irradiated to the fine particles S existing in the vicinity of the condensing point P. In FIG. 2, in order to help understand the principle of the optical tweezers, the fine particles S are illustrated much larger than the actual size.

  The condensed light beam L11 irradiated to the fine particles S is reflected on the surface of the fine particles S or refracted inside the fine particles S, and the traveling direction thereof changes. As a result, the momentum of the condensed light beam L11 changes, a radiation pressure corresponding to the change in the momentum is generated, and a force F as shown by a thick solid line arrow in FIG.

  Here, when the fine particle S is a spherical fine particle having a refractive index larger than the refractive index of the surrounding medium (not shown in FIG. 2) B and having no light absorption, a change in the momentum of the condensed light beam L11. From the above analysis, it is known that the radiation pressure acts on the higher light intensity, and the trapping force that attracts the fine particles S toward the condensing point P acts as the force F. Therefore, in the optical tweezers using the immersion objective lens 6 of the present embodiment, the capturing force F is used to capture and operate the fine particles S. Further, when capturing and operating the fine particles S in the medium B, the state of the fine particles S is observed using an observation system.

  Note that, when the wavelength of the trap light and the wavelength of the observation light are substantially different as in the microscope having the immersion objective lens 6 of the present embodiment described above, it is caused by the longitudinal chromatic aberration of the immersion objective lens 6. However, there is a problem that the position of the condensing point P of the trap light and the in-focus position of the immersion objective lens 6 in the observation system are shifted in the optical axis AX direction, making it difficult to satisfactorily observe the image of the fine particles S. In the microscope having the immersion objective lens 6 of the present embodiment, the position of the condensing point P of the trap light is moved to the optical axis AX by moving the collector lens 3 along the optical axis AX by the action of the drive unit 5. Can move along. With this configuration, the above problem can be solved.

  Further, as described above, the immersion objective lens 6 of the present embodiment is not limited to being used in a transmission optical microscope, but can also be used in an epi-illumination fluorescent microscope, a total reflection illumination fluorescent microscope, etc. The maximum NA of the immersion objective lens 6 is higher than the refractive index of water 1.33, for example, the maximum NA is 1.45. When the microscope having the immersion objective lens 6 is a fluorescence microscope, the transmission optical microscope having the above configuration includes a filter turret having a plurality of filter cubes for fluorescence observation, an epi-illumination light source, and an epi-illumination shutter. Further, a member such as a transmission illumination shutter is added.

  Next, the dichroic film 7 provided on the immersion objective lens 6 of this embodiment will be described.

  For example, when the immersion objective lens 6 having a maximum NA of 1.45 is not provided with the dichroic film 7 having the following characteristics, light having a NA higher than the refractive index of water is trapped as described above. This causes total reflection at the interface between the cover glass and water (medium B) and generates an evanescent field near the interface, resulting in an adverse effect on the performance of the optical tweezers. In order to solve this problem, the immersion objective lens 6 of the present embodiment is provided with a dichroic film 7 deposited on the plane parallel plate 8 in the vicinity of the rear focal plane of the immersion objective lens 6.

FIG. 3 is a view of the dichroic film 7 viewed from above in FIGS. As shown in FIG. 3, the dichroic film 7 has a hollow donut shape, acts only on NA0 or more NA rays that satisfy the following conditional expression (1), and has a λ0 or less that satisfies the following conditional expression (2): It has the characteristic of transmitting only light of wavelength.
(1) na0 ≧ 1.3
(2) λ0 ≦ 800nm

  The NA of the light beam reaching the upper surface of the plane-parallel plate 8 on which the dichroic film 7 is deposited, that is, the light beam reaching the upper surface of the plane-parallel plate 8 from the opposite side of the immersion objective lens 6 (from above in FIG. Trap light, epi-illumination light / excitation light for excitation light for fluorescence observation), or light reaching the upper surface of the plane-parallel plate 8 from the immersion objective lens 6 side (from below in FIG. 2) (for example, the above-mentioned transmitted light) In FIG. 3, the NA of the fluorescent light) is a point on the XY plane (the upper surface of the parallel plane plate 8) formed by the X axis and the Y axis that share the origin coincident with the optical axis AX and are orthogonal to each other. This can correspond to the distance from the origin.

  The dichroic film 7 is configured so that the NA of the light beam reaching the inner peripheral portion becomes na0, and the NA of the light beam reaching the outer peripheral portion (or the inner peripheral portion when the outer diameter is provided with a margin) is liquid. Since it is formed in an annular shape so as to be equal to or greater than the maximum NA (for example, 1.45) of the immersion objective lens 6, it acts only on a light beam having an NA of na0 or more. The transmission wavelength region of the dichroic film 7 is λ0 or less. These na0 (the lower limit value of the NA of the light beam acting on the dichroic film 7) and λ0 (the upper limit value of the wavelength of the light beam transmitted through the dichroic film 7) are arbitrary values satisfying the conditional expressions (1) and (2), respectively. Can be set to For example, na0 = 1.3 and λ0 = 800 nm.

  According to the immersion objective lens 6 of the present embodiment, the above-described trap light, for example, YAG laser light having a wavelength of 1064 nm and longer than λ0 (for example, 800 nm) has a dichroic film in which the NA exceeds na0 (for example, 1.3). 7, an evanescent field due to a trap light component having an NA higher than the refractive index of water is not generated at the interface between the sample cover glass and water (medium B). There is no problem that extraneous external forces work to adversely affect measurement and capture operations. Further, the trap light component having NA smaller than na0 passing through the hollow portion of the dichroic film 7 is irradiated to the fine particles S without being disturbed.

  Further, since the wavelength of visible transmitted illumination light for observing captured fine particles and biological samples, visible excitation light for performing fluorescence observation, and fluorescence from the sample are shorter than λ0, the dichroic film 7 is combined with light passing through the hollow portion. The outer high NA light beam is also transmitted. Therefore, even when the dichroic film 7 is provided, observation similar to that of a normal immersion objective lens can be performed.

  Further, in the present embodiment, even when performing the total reflection fluorescence observation (TIRF) of the objective illumination method, the visible excitation light having an NA higher than the refractive index of water 1.33 passes through the dichroic film 7 having the above characteristics. TIRF can be performed without any problem.

  As described above, according to the immersion objective lens of the present embodiment and the microscope having the immersion objective lens, the occurrence of an evanescent field due to trapped light is prevented without increasing the size and complexity of the apparatus, and optical tweezers. Can improve the performance.

  The above-described embodiment is merely an example, and is not limited to the above-described configuration, and can be appropriately modified and changed within the scope of the present invention.

  For example, the center of the plane parallel plate 8 may be hollow according to the hollow portion of the dichroic film 7.

  Further, the dichroic film 7 can be provided in at least one position near the rear focal plane of the immersion objective lens 6. Alternatively, the dichroic film 7 may be provided not on the immersion objective lens 6 but on a plane conjugate with the rear focal plane.

It is a figure which shows schematically the structure of the microscope which has the immersion objective lens of this embodiment. It is a figure explaining the principle of the optical tweezers using the immersion objective lens of this embodiment. It is a figure which shows the optical member which provided the dichroic film | membrane with which the immersion objective lens of this embodiment is equipped.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 2 Light guide 3 Collector lens 4 Dichroic mirror 5 Drive part 6 Immersion objective lens 7 Dichroic film 8 Parallel plane plate 11 Transmission illumination light source 12 Illumination optical system 13 Second objective lens 14 Image surface 15 Imaging means 16 Eyepiece lens AX Light Axis B Medium S Fine particle P Condensing point F Capture force

Claims (4)

In an immersion objective with NA higher than the refractive index of water,
An immersion objective lens comprising an optical member provided with a dichroic film that acts only on NA0 or more NA rays that satisfy the following conditions and transmits only rays having a wavelength of λ0 or less that satisfy the following conditions.
na0 ≧ 1.3
λ0 ≦ 800nm   The immersion objective lens according to claim 1, wherein the optical member provided with the dichroic film is provided on a rear focal plane of the immersion objective lens or in the vicinity thereof.   The immersion objective lens according to claim 1, wherein the optical member provided with the dichroic film is a plane parallel plate.   A microscope comprising the immersion objective lens according to any one of claims 1 to 3.

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