JPH05188299A - Vertical fluorescent microscope - Google Patents

Abstract

PURPOSE:To provide the vertical fluorescent microscope which can adjust the spectral transmittance characteristics of an excitation filter and absorption filter without exchanging a fluorescent filter set. CONSTITUTION:The excitation filter 16 and absorption filter 20 of the fluorescent filter set 21 are constituted of interference filters consisting of multilayered dielectric films. The excitation filter 16 and absorption filter 20 are constituted to have interlocking mechanisms 33, 35, 36 which support these filters respectively freely turnable with the axis perpendicular to the optical axis and further interlock the turning of the excitation filter 16 and the absorption filter 20.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an epi-fluorescence microscope used for observing biological tissues and cells in the fields of medicine, biology and the like, and more particularly to an epi-fluorescence microscope for adjusting the excitation wavelength of a sample. ..

[0002]

2. Description of the Related Art Generally, an epifluorescence microscope is widely used in fields such as medicine and biology for the purpose of detecting fluorescently labeled proteins and genes on biological tissues and cells.

In particular, in recent years, various fluorescent dyes have been developed to investigate the positional relationship between specific substances and the localization of specific substances in cells by multiple staining using various substances as fluorescent labels. Is used for.

By the way, when observing various fluorescence-stained specimens using such an epifluorescence microscope, a fluorescence filter set having one excitation filter, one dichroic mirror, and one absorption filter is provided for each specific fluorescent dye. It was prepared in advance, and a specific fluorescent dye was observed while changing the excitation and absorption wavelength regions while switching the fluorescence filter set.

[0005]

As described above, in the conventional epi-illumination fluorescence microscope, since the excitation and absorption wavelength regions are fixed for each fluorescence filter set, it is optimal for various fluorescence-stained specimens. In order to observe in the excitation and absorption wavelength regions, a huge number of fluorescent filter sets are required, and there is a problem that the number of parts increases and the cost increases.

If there is a manufacturing error in the excitation filter, the absorption filter, or the dichroic mirror constituting the fluorescence filter set, or if the condition of the fluorescence-stained sample does not match the designed wavelength condition, such Since it was not possible to flexibly adjust the setting conditions of the fluorescence filter in response to the change, there was a problem that the original performance of the fluorescence microscope could not be fully exerted and the observed image deteriorated.

Further, in a fluorescent dye in which the excitation wavelength region and the fluorescence wavelength region are very close to each other, the fluorescence observed depends on where the cutoff wavelength of the spectral transmittance characteristics of the excitation filter and the absorption filter is set. The appearance of the image is very different. Therefore, conventionally, it has been necessary to select a combination of an excitation filter and an absorption filter for a specific fluorescent dye before observing, and the operation is complicated.

The present invention has been made in view of the above circumstances, and sets the spectral transmittance characteristics of the fluorescence filter set to the optimum conditions according to the fluorescent dye of the sample without exchanging the excitation filter and the absorption filter. Epi-fluorescence microscope that can be adjusted and can observe multiple fluorescently stained specimens with one fluorescent filter set, and that the specimens can always be observed under optimal conditions without being affected by the manufacturing error of the fluorescent filter set and the condition of the specimen. The purpose is to provide.

[0009]

In order to achieve the above object, the present invention transmits a light beam emitted from an epi-illumination light source through an excitation filter having a high transmittance in the excitation wavelength region of a fluorescence-stained sample. Let the excitation light, the excitation light is reflected by a dichroic mirror arranged on the observation optical axis and projected on the sample, and the fluorescence emitted from the sample is transmitted through the dichroic mirror and the fluorescence wavelength of the sample. In an epi-fluorescence microscope for observing a fluorescence image of the sample by transmitting an absorption filter having a high transmittance in a region, the excitation filter and the absorption filter are each configured by an interference filter made of a dielectric multilayer film, The excitation filter and the absorption filter are rotatably supported with respect to an axis orthogonal to the optical axis, and the excitation filter and the absorption filter are It was assumed to be engaged by interlocking mechanism the rotation of the filter.

[0010]

According to the present invention, by taking the above means, the excitation filter and the absorption filter can be interlocked with each other through the interlocking mechanism and can be tilted by a predetermined amount with respect to the optical axis. When the excitation filter and the absorption filter are tilted, for example, from a state perpendicular to the optical axis, the incident angle of light on both filters gradually increases. As the angle of incidence increases, the maximum transmittance wavelength of the filter decreases, and the maximum transmittance region gradually shifts to the short wavelength side.

Therefore, the maximum transmittance wavelengths of the excitation filter and the absorption filter are set to appropriate wavelengths, and the excitation filter and the absorption filter are rotated according to the fluorescent dye of the sample to replace the fluorescence filter set. Without this, it is possible to adjust the optimum excitation and absorption wavelength range.

[0012]

Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 shows the configuration of an optical system of an epi-illumination fluorescence microscope according to the first embodiment of the present invention, and FIG. 2 shows a fluorescent filter set provided in the optical system.

In the epi-illumination fluorescence microscope of this embodiment, a light flux emitted from an epi-illumination light source 11 such as a mercury lamp is condensed by a collector lens 12 and is passed through an aperture diaphragm 13, a field diaphragm 14 and a collector lens 15. , Enters the excitation filter 16 having the spectral transmittance characteristic shown by the solid line E in FIG. The light flux that has passed through the excitation filter 16 becomes excitation light having a predetermined excitation wavelength, is incident on the dichroic mirror 17 that is arranged on the observation optical axis and has the spectral transmittance characteristic shown by the broken line DM in FIG. Is reflected in.
The excitation light that has entered the observation optical axis passes through the objective lens 18 and is projected onto the sample S. The sample S is placed on a stage 19 that can move up and down.

The sample S illuminated by the excitation light is
Fluorescence is emitted on the longer wavelength side than the excitation wavelength. The fluorescence from the sample S having such a wavelength characteristic is incident on the objective lens 18, further transmitted through the dichroic mirror 17,
The light enters the absorption filter 20 having the spectral transmittance characteristic shown by the chain line B in FIG.

That is, by setting the spectral transmittance characteristic of the absorption filter 20 according to the wavelength characteristic of the fluorescence emitted from the sample S, only the fluorescence is transmitted through the absorption filter 20. Excitation filter 16, dichroic mirror 17, absorption filter 20 to fluorescence filter set 2
Make up one.

The fluorescence of the sample S transmitted through the absorption filter 20 enters the prism 22 arranged on the observation optical axis, and the observation optical system having the eyepiece lens 23 and the photographing lens 24.
The optical path is branched to the photographing optical system in which is arranged.

As shown in FIG. 2, the fluorescent filter set 21 includes a fluorescent cube 30 having a cylindrical through hole at the center and an opening communicating with the through hole on one side surface. The excitation filter 16 is arranged at the opening of the absorption filter 20, and the absorption filter 20 is provided at one end of the through hole.
Further, the dichroic mirror 17 is arranged at an angle of 45 degrees in the through hole and at a position facing the opening. Further, on the other side surface of the fluorescent cube 30, a mounting portion 31 for detachably mounting on the microscope body is formed.

Excitation filter 16 and absorption filter 20
Is an interference filter made of a dielectric multilayer film. The excitation filter 16 has a rotation axis 3 perpendicular to the optical axis.
The rotating shaft 32 is supported by the fluorescent cube 30. This allows the excitation filter 16 to rotate about an axis perpendicular to the optical axis. Rotating shaft 32
A gear 33 rotatably supported by the fluorescent cube 30 is attached to one end of the.

The absorption filter 20 is supported by a rotary shaft 34 which is perpendicular to the optical axis and is supported by the fluorescent cube 30. As a result, the absorption filter 20 is also rotatable about the axis perpendicular to the optical axis. A gear 35 rotatably supported by the fluorescent cube 30 is attached to one end of the rotary shaft 34.

The gear 33 of the excitation filter 16 and the gear 35 of the absorption filter 20 are connected via a gear 36 rotatably supported by the fluorescent cube 30 to form an interlocking mechanism.

Therefore, in the excitation filter 16 and the absorption filter 20, the angle (incident angle of the light beam) with the optical axis changes in conjunction with each other. The gear reduction ratios of the gears 33, 35, 36 are set so that the shift amounts of the cutoff wavelengths caused by tilting the excitation filter 16 and the absorption filter 20 with respect to the optical axis are the same. Here, the relationship between the change in the incident angle to the excitation filter 16 and the absorption filter 20 and the maximum transmittance region will be described.

Generally, the interference condition of the interference filter is that the maximum transmittance wavelength is λ, the optical thickness of the dielectric (including the phase difference generated at the boundary of the dielectric is also included in the optical path length), and the boundary. When the angle of reflection by φ is φ, it can be expressed by the following relational expression. 2t · cos φ = mλ

Here, when the order m is constant and the interference condition is constant, the wavelength λ is proportional to cos φ. This φ
Is the reflection angle, but it is considered to be equivalent because it is conjugate with the incident angle according to Snell's law. This means that if the incident angle is increased, cos φ will decrease, and at the same time, the wavelength λ will also decrease, whereby the maximum transmittance region will gradually shift to the short wavelength side. ..

Therefore, when the incident surfaces of the excitation filter 16 and the absorption filter 20 are gradually inclined from the state perpendicular to the optical axis, the spectral transmittance characteristics E and B of the excitation filter 16 and the absorption filter 20 are as shown in FIG. As shown in FIG. 4, E, B are shifted to E ′, B ′ by Δλ to the short wavelength side.

In the present embodiment configured as described above, by rotating any one of the gears 33, 35 and 36, the remaining two gears also rotate in conjunction via the gear 36, The excitation filter 16 and the absorption filter 20 that are integrated with 33 and 35 rotate about the rotation shafts 32 and 34, respectively.

As a result, the incident angle of each light beam entering the excitation filter 16 and the absorption filter 20 changes, and the spectral transmittance characteristics E and B of the excitation filter 16 and the absorption filter 20 shift as described above.

Therefore, when there is a manufacturing error in the excitation filter 16 and the absorption filter 20 and the dichroic mirror 17 which constitute the fluorescence filter set, or when there is a change in the optimum excitation state depending on the state of the sample S, as described above. By rotating the excitation filter 16 and the absorption filter 20 to adjust the spectral transmittance characteristics E and B to the optimum states, the sample S is always kept in the optimum excitation state without exchanging the fluorescence filter set. The fluorescent image can be observed. Therefore, it is possible to reliably prevent the deterioration of the fluorescence image due to the mismatch between the spectral transmittance characteristics of the excitation filter 16 and the absorption filter 20 and the fluorescent dye, and it is possible to improve the observation ability.

Further, since the spectral transmittance characteristics of the excitation filter 16 and the absorption filter 20 can be continuously changed with one fluorescence filter set 21, the excitation switching operation becomes unnecessary and the operability can be improved. Moreover, since the spectral transmittance characteristics can be continuously changed, one fluorescent filter set 21 can be adjusted to the optimum excitation condition for more samples, and as a result, the number of parts and the cost can be reduced. it can. Hereinafter, various modifications of the configuration related to the fluorescence filter set will be described with reference to FIGS. 5 to 9.

In the first modified example shown in FIG. 5, four types of fluorescent filter sets 41 to 44 of different types are held by a turret (not shown) having a rotation axis O parallel to the observation optical axis, and arbitrary. The above fluorescence filter set can be inserted into and removed from the observation optical path. FIG. 6 shows a fluorescent filter set 41.
Is shown in the cross section in the state of being arranged on the observation optical path.

The fluorescent filter sets 41 to 44 have different spectral transmittance characteristics, and their individual structures are almost the same as the fluorescent filter set 21 described in the above-mentioned embodiment. That is, the excitation filter 16 is rotatably arranged around the rotation shaft 32 around the rotation axis 32 in the opening on the light source side of the fluorescent cube 30, and the absorption filter 20 is rotated in the prism-side opening of the through-hole of the fluorescence cube 30. It is arranged rotatably around the shaft 34 together with the rotary shaft. Gears 33 and 35 are concentrically fixed to the respective ends of the rotary shaft 32 and the rotary shaft 34 extending from the side surface of the fluorescent cube 30, and the gears 33 and 35 are connected by a gear 36.

According to the modified example thus constructed, the spectral transmittance characteristics of the excitation filter 16 and the absorption filter 20 can be continuously changed by the individual fluorescent filter sets 41 to 44, and the emission wavelength of the fluorescent dye can be changed. In the case of a large difference, the turret can be rotated to arrange a fluorescent filter set having an appropriate spectral transmittance characteristic on the optical path to obtain optimum excitation conditions.

In the second modification shown in FIGS. 7 and 8, three types of fluorescent filter sets 51 to 53 of different types can be inserted into and removed from the observation optical path, and the excitation filter 1 of each fluorescent filter set can be used. The absorption filter is configured to be linked by belt transmission. In this modification, a guide member 54 is fixed at a position apart from a position where the fluorescence filter set is to be arranged, and a surface of the guide member 54 facing the observation optical axis is orthogonal to the observation optical axis. Is formed with a guide groove. This guide member 54
A slider 55 is attached to the guide groove by slidably fitting the linear guide portion 55a into the guide groove. On the other surface of the slider 55, the above three types of fluorescent filter sets 51 to 53 are attached and fixed at equal intervals.

The fluorescent filter sets 51 to 53 have the same individual structure except that the spectral transmittance characteristics are different. Therefore, the structure of the fluorescent filter set 51 will be described as an example. The fluorescent cube 56 has the same shape as that described in the first embodiment, and the excitation filter 16 is integrated with the rotating shaft 57 in the opening on the light source side and rotates about the rotating shaft 57. Being supported to move. A pulley 58 is attached to this rotating shaft 57.

The absorption filter 20 is supported by an opening on the prism side of the through-hole of the fluorescent cube 56 so as to rotate integrally with the rotating shaft 59 about the rotating shaft 59. A pulley 60 is attached to the.
A belt 61 is hung between the two pulleys 58 and 60.

Here, the diameter ratio of the pulleys 58 and 60 is set so that the shift amounts of the cutoff wavelengths caused by tilting the excitation filter 16 and the absorption filter 20 with respect to the optical axis are the same.

In the modified example having such a configuration, the slider 55 is moved along the guide member 54 in the direction orthogonal to the observation optical axis so that the desired fluorescence filter set (51, 52, 53) can be observed. Located on the street.

When it is necessary to adjust the spectral transmittance characteristics of the excitation filter 16 and the absorption filter 20, the belt 61 is driven by a drive mechanism (not shown).
As a result, the excitation filter 16 and the absorption filter 20 are rotated in conjunction with each other, and the spectral transmittance characteristics of each are shifted in a predetermined direction, so that optimum conditions can be set. In the third modification shown in FIG. 9, the excitation filter 16 and the absorption filter 20 are
It is tilted in conjunction with each other by being electrically driven.

In this modification, the fluorescent cube 60 is provided with step motors 62a and 62b for independently rotating and driving the excitation filter 16 and the absorption filter 20 of the fluorescence filter set.

Then, the memory 63 stores the respective rotation angles at which the cutoff wavelength shift amounts of the excitation filter 16 and the absorption filter 20 become the same. Also encoder 64
When the excitation wavelength shift signal is input to the CPU 65 from the above, the interlocking circuit 66 causes the excitation filter 16 and the absorption filter 20 to have the same cutoff wavelength shift amount according to the shift amount indicated by the excitation wavelength shift signal. Is configured to calculate.

By outputting the calculation result by the interlocking circuit 66 to the drivers 67 and 68 to drive the step motors 62a and 62b, the excitation filter 16 and the absorption filter 20 can be interlocked and tilted by a desired amount.

In this modification, since the rotation angle data is stored in the memory 63, even if the interlocking relationship between the excitation filter 16 and the absorption filter 20 is not constant, any rotation angle data can be set according to the rotation angle data. Control becomes possible. The present invention is not limited to the above-described one embodiment and each modified example, and various modifications can be made without departing from the gist of the present invention.

[0042]

As described in detail above, according to the present invention, the spectral transmittance characteristics of the fluorescence filter set are
You can adjust the optimum conditions according to the fluorescent dye of the sample without changing the absorption filter, you can observe multiple fluorescently stained samples with one fluorescent filter set, and also the manufacturing error of the fluorescent filter set and the condition of the sample It is possible to provide an epi-illumination fluorescence microscope that can always observe under optimum conditions without being affected by.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an optical system of an epi-illumination fluorescence microscope according to an embodiment of the present invention.

FIG. 2 is a cross-sectional view of a fluorescence filter set included in an epi-illumination fluorescence microscope according to an embodiment.

FIG. 3 is a diagram showing a spectral transmittance characteristic of the fluorescence filter set shown in FIG.

FIG. 4 is a diagram showing that the spectral transmittance characteristics are shifted by operating the fluorescence filter set shown in FIG.

FIG. 5 is a diagram showing a first modification of a portion related to the fluorescence filter set.

FIG. 6 is a sectional view of the first modified example.

FIG. 7 is a diagram showing a second modification of the portion related to the fluorescence filter set.

FIG. 8 is a sectional view of the second modified example.

FIG. 9 is a diagram showing a third modification of the portion related to the fluorescence filter set.

[Explanation of symbols]

11 ... Light source, 16 ... Excitation filter, 17 ... Dichroic mirror, 20 ... Absorption filter 21, 41-44, 5
1 to 53 ... Fluorescent filter set, 33, 35, 36 ... Gear, 58, 60 ... Pulley, 61 ... Belt, 62a, 62
b ... Step motor.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takayuki Shimizu 2-43-2 Hatagaya, Shibuya-ku, Tokyo Inside Olympus Optical Co., Ltd.

Claims (1)

[Claims] 1. A light flux emitted from an epi-illumination light source,
The excitation light having a high transmittance in the excitation wavelength region of the fluorescence-stained specimen is excited light, and the excitation light is reflected on a dichroic mirror arranged on the observation optical axis and projected onto the specimen, And in the epi-illumination fluorescence microscope for observing the fluorescence image of the sample by transmitting the fluorescence emitted from the sample through the dichroic mirror and the absorption filter having a high transmittance in the fluorescence wavelength region of the sample, the excitation filter and Each of the absorption filters is formed of an interference filter made of a dielectric multilayer film, and the excitation filter and the absorption filter are rotatably supported with respect to an axis orthogonal to the optical axis. An epi-fluorescence microscope characterized in that its rotation is interlocked by an interlocking mechanism.