JP4454980B2 - Microscope imaging optical system and microscope using the same - Google Patents

Description

The present invention relates to an imaging optical system of a microscope and a microscope using the same , and in particular, splits light from a specimen according to optical characteristics such as a wavelength, and projects each to different positions in an imaging plane , and The present invention relates to a microscope using the same .

  Conventionally, in the research field for analyzing physical properties of specimens such as living cells, different tissues in cells are labeled with reagents having different fluorescence wavelengths, and observation by multiwavelength excitation and multiwavelength photometry is generally performed. Also, in recent years, as a means to ensure the temporal identity of specimen images by multiwavelength photometry and the sensitivity of the imaging device, the angle of view of one imaging device is divided into a plurality of regions, and optical characteristics such as wavelength An apparatus equipped with an imaging optical system configured to project a projection image of a specimen separated for each of the specimens onto different regions has been devised, and widely used, for example, as shown in Patent Document 1 and Patent Document 2 It has been. In Patent Document 1, as shown in FIG. 9, in an imaging optical system, light diverging from a point 106 in a sample is converted into parallel rays by an objective lens 110, and then optical characteristics such as wavelength are separated by a separation selection unit 111. In this configuration, the optical axes are separated by the optical axis conversion means 112 and then projected onto different positions A and B within the angle of view of the imaging device through the imaging lens 113.

In Patent Document 2, as shown in FIG. 10, in an imaging optical system, light diverging from a point 200 in a specimen is projected as an intermediate image 218 onto a field stop 220 through lens groups 214 and 216, and the intermediate image is obtained. The light diverging from 218 is converted into parallel rays by a collimator lens 224 and then split by a splitting prism 226. It is configured to project to positions with different corners.
JP-A-5-164687 Special Table 2001-525534

    When projecting a plurality of projection images onto different regions within the angle of view of a single imaging device, it is usual that the focal position of each projection image is configured to match on the imaging surface of the imaging device. . In the imaging devices according to Patent Document 1 and Patent Document 2, the observation light path is divided in parallel light beams, and each parallel light beam is collected by the same imaging lens. The focal positions of the images coincide with each other on the imaging surface. On the other hand, the observation target in the field of living cell research is not limited to a specific height surface in the specimen, and there are cases where it is desired to observe simultaneously, for example, phenomena occurring inside the cell and in the vicinity of the cell membrane. By using an imaging optical system that divides and projects an image observed with an objective lens into different positions within the angle of view of the imaging device, information of different heights on the specimen can be simultaneously imaged with a single imaging device. If possible, observation, detection, measurement, and the like of temporal identity can be realized, and only one imaging device is required and the device is excellent in terms of cost. However, in the imaging apparatuses shown in Patent Document 1 and Patent Document 2, as described above, the focal positions of the divided projection images are fixed so as to coincide with each other on the imaging surface. Is not considered.

The present invention has been made in view of the above problems, and an imaging optical system capable of dividing and projecting surfaces with different heights of a specimen on an imaging surface of an imaging apparatus and simultaneously observing the imaging surface An object is to provide a microscope using the same .

In order to achieve the above object, an imaging optical system of a microscope according to the present invention includes an objective lens that guides light from a specimen to an observation optical path that reaches an image plane, and a parallel light flux emitted from the objective lens toward the image side or convergent. an imaging lens for converting the light beam into a convergent light beam for focusing on the imaging surface, the optical characteristics of the wavelength, such as a convergent light bundle converted by the imaging lens the imaging lens be disposed on the image side of the dividing means for dividing, and the total reflection means a group of the projected image of the converging light beam of each divided are arranged to form at different positions within the imaging plane by said dividing means, divided by the previous SL dividing means anda optical path length conversion means for moving the focal position of the converging light beam by increasing or decreasing the air conversion value of at least one of the optical path length or optical path length of converging light bundle of each that is, different on the specimen Surface of height Characterized by simultaneously observed.

Further, the microscope imaging optical system of the present invention, before Kihikariro length conversion means, and characterized by being constituted by a moving means for integrally moving the total reflection surface of the at least two positions of the total reflection means group To do.
The imaging optical system of the microscope of the present invention includes excitation light illumination means capable of simultaneous illumination with a plurality of excitation wavelengths including at least one total reflection fluorescence illumination means, and is excited by the total reflection fluorescence illumination means. Is picked up either on the optical path side including the optical path length converting means or on the other optical path side.
In addition, the imaging optical system of the microscope of the present invention includes a light shielding unit for preventing an overlap between the projection image of the first convergent beam bundle and the projected image of the second convergent beam bundle divided by the dividing unit. It is characterized by that.

In order to achieve the above object, the microscope of the present invention includes an objective lens that guides light from a specimen to an observation optical path that reaches an image plane, and a parallel ray bundle or a convergent ray bundle emitted from the objective lens to the image side. an imaging lens for converting the convergent light flux condensed by the imaging plane, to divide the convergent light beam, wherein they are arranged on the image side of the imaging lens is converted by the imaging lens by the optical characteristics of the wavelength, etc. dividing means, and the total reflection means group arranged the projection image of the convergent light beam of each divided to form at different positions within the imaging plane by said dividing means, divided by the previous SL dividing means Optical path length conversion means for moving the focal position of the convergent ray bundle by increasing or decreasing the optical path length of at least one of the convergent ray bundles or an air-converted value of the optical path length, and each projection image formed at the different positions Before Anda imaging device for imaging the imaging surface, characterized by observing the surface of different heights on the specimen at the same time.

  As described above, according to the present invention, it is possible to simultaneously observe and image surfaces of different heights on a specimen with a single imaging device, and it is possible to provide a cost-effective device.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 is a conceptual diagram of the overall configuration of the first embodiment of the imaging optical system of the microscope of the present invention. The symbols used in the description of the present embodiment are commonly used in the description of the other embodiments.

  In FIG. 1, 1 is a sample, 2 is an objective lens that guides the light from the sample 1 to the observation optical path 0 to the image plane, and 3 is a parallel beam bundle emitted from the objective lens 2 and converted into a convergent beam bundle. An image forming lens 4 is a reflecting means 4 constituted by a mirror or a prism for reflecting the convergent light beam emitted from the image forming lens to the image pickup apparatus side. Reference numeral 5 denotes a dichroic mirror that selectively separates the convergent light beam into transmission and reflection according to wavelength. Here, light having a wavelength longer than a specific wavelength set by the dichroic mirror 5 is transmitted to form a first convergent light bundle 01, and light having a short wavelength is reflected to form a second convergent light bundle 02. To do. Reference numerals 61 and 62 denote a first filter and a second filter having a characteristic of selectively transmitting only the wavelength band used for each observation in the first convergent light beam 01 and the second convergent light beam 02, respectively. It is. Reference numeral 7 denotes a first total reflection means composed of a total reflection prism that reflects the first convergent beam bundle 01 transmitted through the dichroic mirror 5, and 8 denotes a second convergent beam bundle 02 reflected from the dichroic mirror 5. This is a second total reflection means composed of a total reflection mirror that totally reflects to the imaging device 9 side. 10 totally reflects the first convergent beam bundle 01 reflected by the first total reflection means 7 toward the image pickup device 9, and the second convergent beam bundle 02 reflected by the second total reflection means 8 is on its side. It is the 3rd total reflection means arrange | positioned in the position which passes through. The first total reflection unit 7, the second total reflection unit 8, and the third total reflection unit 10 convert the projection images of the first convergent ray bundle 01 and the second convergent ray bundle 02 into the imaging device 9. Are arranged at different positions within the angle of view. Reference numeral 13 denotes a stop for preventing the projection image of the first convergent beam bundle 01 and the projection image of the second convergent beam bundle 02 from overlapping within the angle of view of the imaging device 9. Reference numeral 11 denotes an optical path length conversion means provided between the first total reflection means 7 and the third total reflection means in the first convergent light beam 01. The optical path length conversion means 11 is composed of a fixed wedge prism 11a and a moving wedge prism 11b, and the fixed wedge prism 11a and the moving wedge prism 11b have the same wedge angle, and the surfaces facing each other are parallel and opposite to each other. Are arranged so as to be perpendicular to the optical axis of the first convergent light beam 01. The moving wedge prism 11b is provided so as to be movable in a direction parallel to the surface facing the fixed wedge prism 11a. Incidentally, the total of the glass path lengths of the total reflection prism used in the first total reflection means 7 and the wedge prisms 11a and 11b used in the optical path length conversion means 11 is the first convergent light beam 01 and the second convergent light beam. The focal position of the bundle 02 is set so as to substantially match in the imaging device 9.

  Next, the operation and effect of the present embodiment will be described. 2 (a) and 2 (b) show a state in which the moving wedge prism 11b is moved in the optical path length conversion means 11. FIG. In FIG. 2, when the moving wedge prism 11b is moved from the state of FIG. 2 (a) to the state of FIG. 2 (b), the glass of the first convergent beam bundle 01 passing through the fixed wedge prism 11a and the moving wedge prism 11b. The road length changes. The same applies to the case where the moving wedge prism 11b is moved from the state shown in FIG. 2B to the state shown in FIG. Here, the distance between the fixed wedge prism 11a and the movable wedge prism 11b is always constant, and the angle at which the optical axis of the first convergent beam bundle 01 is incident on the fixed wedge prism 11a and the angle at which it is emitted from the movable wedge prism 11b is Since it is always vertical, the optical axis of the first convergent light beam 01 does not change. When the glass path length in the optical path length conversion means 11 increases, the air-converted value of the optical path length decreases, so that the focal position of the first convergent light beam 01 moves further past the imaging device 9. Conversely, when the glass path length in the optical path length conversion means 11 decreases, the air-converted value of the optical path length increases, so that the focal position of the first convergent beam bundle 01 moves to the front of the imaging device 9.

Next, a case where the moving wedge prism 11b is intentionally shifted will be described with reference to FIG. In FIG. 3, the projected image of the surface 1 a on the specimen 1 at the focal position of the objective lens 2 is projected onto the imaging device 9 by the convergent light bundle 02. On the other hand, when the air wedge value of the optical path length of the convergent beam bundle 01 is changed by intentionally shifting the moving wedge prism 11b, the focal position of the convergent beam bundle 01 moves. On the other hand, for example, when the focal position of the convergent beam bundle 01 is moved forward by a distance X from the imaging device 9, and the image of the sample surface 1a is projected at this position, it is on the imaging device 9 in this state. When the image point is back-projected on the objective lens 2 side, the ray bundle passes through an optical path as indicated by a dotted line 03, and the distance X / M ² (M is the distance of the objective lens 2) from the specimen surface 1a which is the focal plane of the objective lens 2. (Magnification) is projected onto 1b close to the objective lens. In other words, in the observation optical path passing through the dichroic mirror 5 and passing through the optical path length conversion means 11, an image of the sample surface 1 b at a position closer to the focal position of the objective lens 2 by the distance X / M ² is on the imaging device 9. Projected.

As described above, according to the configuration of the present embodiment, it is possible to project projected images of different heights on the specimen 1 at different positions within the angle of view of the imaging device 9 and simultaneously observe them. . For example, if a 40x objective lens is used and the objective lens 2 is focused at a position 1 μm inside the cell adhered to the cover glass, the optical path length on the image side is increased by 0.001 × 40 ² = 1.6 mm. Thus, the cell adhesion surface of the cover glass can be projected. According to the configuration of the present embodiment, this can be realized by reducing the glass path length in the optical path length conversion means 11 by 4.8 mm (when the refractive index of the glass used is 1.5).

  As described above, the first embodiment of the present invention has been described. As a modified example, instead of continuous correction means such as the optical path length conversion means 11, several parallel plane glass plates with different thicknesses are selectively used. A similar result can be obtained by inserting into the convergent beam. The light splitting means is not limited to the dichroic mirror, and a half mirror or a polarization beam splitter may be used.

  FIG. 4 is a conceptual diagram of the overall configuration of the second embodiment of the present invention. Components identical to those in FIG. Description of these will be omitted.

  In FIG. 4, reference numeral 12 denotes a fourth total reflection beam configured to totally reflect the first imaging light beam 01 reflected by the first total reflection unit 7 twice and guide it to the third total reflection unit 10. It is a reflection means. Here, the fourth total reflection means 12 is configured such that the optical axis of the first convergent beam bundle 01 is parallel on the incident side and the emission side, and the fourth total reflection means 12 itself is the first total reflection means 12. It is provided so as to be movable in a direction parallel to the optical axis on the incident side and the exit side of the convergent light beam. Reference numeral 14 denotes a transmission prism for matching the air-converted value of the optical path length of the first imaging beam bundle 01 with the second imaging beam bundle.

Next, the operation and effect of the present embodiment will be described. FIG. 5 shows the states (a) and (b) in which the fourth total reflection means 12 is moved.
As can be seen from FIGS. 5A and 5B, when the fourth total reflection means 12 is moved in the direction of the arrow, the glass path length of the first convergent light beam 01 changes. Here, the moving direction of the fourth total reflection means 12 is parallel to the incident-side and exit-side optical axes of the first convergent beam bundle 01, so the optical axis of the first convergent beam bundle 01 does not change. . As shown in FIG. 5A, when the fourth total reflection means 12 is brought closer to the first total reflection means 7, the optical path length is reduced, so that the focal position of the first convergent beam bundle 01 passes through the imaging device 9. Move ahead. On the other hand, as shown in FIG. 5B, when the fourth total reflection means 12 is moved away from the first total reflection means 7, the optical path length increases, so that the focal position of the first convergent light beam 01 is increased. Moves in front of the imaging device 9.

Next, the case where the fourth total reflection means 12 is intentionally shifted will be described with reference to FIG. In FIG. 6, the projected image of the surface 1 a on the sample 1 at the focal position of the objective lens 2 is projected onto the imaging device 9 by the convergent light bundle 02. On the other hand, when the optical path length of the convergent light beam 01 is changed by intentionally shifting the fourth total reflection means 12, for example, the fourth total reflection means 12 is moved away from the first total reflection means 7 by a distance X / 2. In this case, the focal position of the convergent light beam 01 moves forward by a distance X from the imaging device 9, and the image of the sample surface 1a is projected at this position. On the other hand, when the image point on the imaging device 9 is back-projected to the objective lens 2 side in this state, the light beam passes through the optical path as indicated by the dotted line 03 and is a distance from the sample surface 1a that is the focal plane of the objective lens 2. It is projected onto 1b close to the objective lens by X / M ² (M is the magnification of objective lens 2). In other words, in the observation optical path that passes through the dichroic mirror 5 and passes through the fourth total reflection means 12, an image of the sample surface 1 b at a position closer to the focal position of the objective lens 2 by a distance X / M ² is obtained. Projected on top.

According to the configuration of the second embodiment, for example, a 40 × objective lens is used at different positions within the angle of view of the imaging device 9, and the focal point of the objective lens 2 is focused at a position of 1 μm inside the cell adhered to the cover glass. When combined, the cell adhesion surface of the cover glass can be projected by increasing the optical path length on the image side by 0.001 × 40 ² = 1.6 mm. According to the configuration of the present embodiment, this can be realized by moving the fourth total reflection means 12 by 0.8 mm.

  FIG. 7 is a conceptual diagram of the overall configuration of the third embodiment of the present invention. The same components as those in FIGS. 1 to 6 are denoted by the same reference numerals. Description of these will be omitted.

  In FIG. 7, 21a is a light source for epi-illumination, and 22a is a projection lens for epi-illumination light. Reference numeral 25a denotes an excitation filter that transmits only a specific wavelength of epi-illumination light. Reference numeral 23a denotes a dichroic mirror that reflects the excitation light transmitted through the excitation filter 25a to the objective lens 2 side. Reference numeral 62 denotes a filter in the second convergent light bundle 02, which has a characteristic of selectively transmitting the fluorescence of the fluorescent reagent excited by light in the wavelength band that has passed through the excitation filter 25a. . Reference numeral 21b denotes a light emitting part for total reflection fluorescent illumination constituted by an emission end of a glass fiber, and 22b denotes a projection lens for total reflection illumination light. Reference numeral 24 denotes a condenser lens disposed at a position facing the objective lens 2 with the sample 1 interposed therebetween, and has a numerical aperture capable of total reflection illumination. Reference numeral 23b denotes a mirror that reflects the total reflection illumination light toward the condenser lens 24 side.

  The total reflection illumination light reflected from the mirror 23b is condensed at a position shifted from the optical axis on the front focal plane of the condenser lens 24, and after exiting the condenser lens 24, the sample 1 has an angle larger than the total reflection angle. Incident and totally reflected. Only evanescent light enters the sample 1 side at a depth of several hundred nm or less and is used as excitation light. On the other hand, as the filter 61 provided in the first convergent light beam 01, a filter having a characteristic of selectively transmitting fluorescence of a fluorescent reagent excited by light in the wavelength band of evanescent light is used.

  Next, the operation and effect of the present embodiment will be described. Of the fluorescence excited by the excitation light of the epi-illumination, a projection image of the fluorescence emitted from the surface 1a on the specimen 1 at the focal position of the objective lens 2 is projected onto the imaging device 9 by the convergent light beam 02. . On the other hand, by shifting the fourth total reflection means 12 to reduce the optical path length of the convergent light beam 01, the position of the sample surface 1b at a position away from the focal position of the objective lens 2, that is, a position close to the condenser lens 24 is obtained. An image is projected on the imaging device 9. When the amount of decrease in the optical path length is adjusted and the sample surface 1b is brought near the cover glass surface on the condenser lens 24 side, excitation light from the evanescent light entering from the condenser lens arrives, so that the sample surface 1b is emitted. The projection image of the fluorescence is projected onto the image pickup device 9 by the convergent light beam 01.

As described above, according to the configuration of the third embodiment, the two-wavelength simultaneous excitation illumination using the epi-illumination and the total reflection illumination is used to observe the detailed shape by the epi-illumination while aligning the focal position of the objective inside the sample. The projected image obtained by the optical path length conversion is matched with the vicinity of the cover glass to detect fluorescence by total reflection illumination, and these can be simultaneously imaged by one imaging device. For example, in the observation of living cells, the observation of Ca ²⁺ -induced protein behavior in the vicinity of the cytoskeleton by epifluorescence and the detection of luminescence associated with the opening emission in cell adhesion spots by total reflection fluorescence are performed simultaneously. It is possible to provide an imaging apparatus that can meet various needs.

  As described above, the third embodiment of the present invention has been described. As a modification, as shown in FIG. 8, the incident illumination light source 21a, the projection lens 22a, and the mirror 23b are provided on the condenser lens 2 side, and evanescent light is emitted. It is also possible to make the total reflection illumination light having two types of wavelengths having different penetration depths incident from the condenser lens 24 side. It is also possible to adopt a configuration in which the total reflection illumination light is incident not from the condenser lens 24 side but from the objective lens 2 side. A similar effect can be achieved by such a configuration.

It is a conceptual diagram of the whole structure of the 1st Example of the imaging optical system of the microscope of this invention. It is a conceptual diagram of the optical path length conversion means in 1st Example of the imaging optical system of the microscope by this invention. (a) is a figure which shows the state which moved the wedge prism, (b) is a figure which shows the state which moved the wedge wedge prism to another position. It is a figure which shows the state which shifted the wedge wedge prism in the structure of 1st Example of the imaging optical system of the microscope by this invention. It is a conceptual diagram of the whole structure concerning the 2nd Example of the imaging optical system of the microscope of this invention. It is a figure which shows the state (a) which moved the total reflection means in the 2nd Example of the imaging optical system of the microscope by this invention, and another state (b). It is a figure which shows the state at the time of deliberately shifting a total reflection means in the 2nd Example of the imaging optical system of the microscope by this invention. It is a conceptual diagram of the whole structure concerning the 3rd Example of the imaging optical system of the microscope of this invention, and its illumination means. It is a conceptual diagram of a structure of the modification concerning 3rd Example of this invention. It is a conceptual block diagram of the conventional imaging optical system. It is a conceptual block diagram of the other conventional imaging optical system.

Explanation of symbols

0, 01, 02, 03 Ray bundle 1 Sample 2 Objective lens 3 Imaging lens 4 Mirror 5 Dichroic mirror 7, 8, 10 Total reflection means 9 Imaging device 11 Optical path length conversion means 11a Fixed wedge prism 11b Moving wedge prism 13 Aperture 14 Transmissive prism 21a Epi-illumination light source 21b Fluorescent illumination beam emitting portions 22a and 22b Projection lenses 23a and 23b Mirror 25a Excitation filters 61 and 62 Filter X Focus movement distance

Claims (5)

An objective lens that guides the light from the specimen to the observation optical path to the image plane;
An imaging lens that converts a parallel beam bundle or a convergent beam bundle emitted from the objective lens to the image side into a convergent beam bundle that is collected on the imaging surface ;
Dividing means for dividing the converging luminous flux is converted be disposed on the image side of the imaging lens by the imaging lens by the optical characteristic of the wavelength, etc.,
A total reflection means group arranged and formed so as to form projected images of the respective convergent beam bundles divided by the dividing means at different positions in the imaging surface ;
An optical path length conversion means for moving the focal position of the converging light beam by increasing or decreasing the air conversion value of at least one of the optical path length or optical path length of converging light bundle of each divided by the previous SL dividing means,
Equipped with,
An imaging optical system of a microscope characterized by simultaneously observing surfaces having different heights on the specimen .   2. The imaging optical system of a microscope according to claim 1, wherein the optical path length conversion unit is configured by a moving unit that integrally moves at least two total reflection surfaces in the total reflection unit group. Including excitation light illumination means capable of simultaneous illumination with a plurality of excitation wavelengths including at least one total reflection fluorescence illumination means, and the fluorescence excited by the total reflection fluorescence illumination means is on the optical path side or the other side including the optical path length conversion means The imaging optical system for a microscope according to claim 1, wherein an image is picked up on any one of the optical path sides of the microscope . 4. A light shielding unit for preventing an overlap between a projected image of the first convergent beam bundle and a projected image of the second convergent beam bundle divided by the dividing unit. An imaging optical system for a microscope according to any one of the above. An objective lens that guides the light from the specimen to the observation optical path to the image plane;
An imaging lens that converts a parallel beam bundle or a convergent beam bundle emitted from the objective lens to the image side into a convergent beam bundle that is collected on the imaging surface;
A dividing unit that is arranged on the image side of the imaging lens and divides the convergent light beam converted by the imaging lens according to optical characteristics such as a wavelength;
A total reflection means group arranged and formed so as to form projected images of the respective convergent beam bundles divided by the dividing means at different positions in the imaging surface;
Optical path length conversion means for moving the focal position of the convergent light bundle by increasing or decreasing the optical path length of at least one of the convergent light bundles divided by the dividing means or an air-converted value of the optical path length;
An imaging device that captures images of the projected images formed at the different positions on the imaging surface;
Comprising
A microscope characterized by simultaneously observing surfaces of different heights on the specimen.

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