Fluorescence detecting device

• US 6,094,274 A
• Filed: 06/04/1999
• Issued: 07/25/2000
• Est. Priority Date: 06/05/1998
• Status: Expired due to Term

• Current Assignee: Olympus Optical Corporation Limited (Olympus Corporation)
• Original Assignee: Olympus Optical Corporation Limited (Olympus Corporation)
• Inventors: Yokoi, Eiji
• Primary Examiner(s): EVANS, FANNIE L
• Application Number:
  US09/325,541
• Time in Patent Office: 417 Days
• Field of Search: 356/317, 356/318, 356/417, 250/458.1, 250/459.1, 250/461.1, 250/461.2, 359/389
• US Class Current: 356/417
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 26/007 the movable or deformable optical element controlling the colour, i.e. a spectral characteristic, of the light

Abstract

The present invention provides a fluorescence detecting device comprising a light source, an excitation filter set, a dichroic mirror, an objective, an absorption filter set and an observation optical system, wherein

the excitation filter set includes two interference films with a transmittance of 25% or more at a wavelength by 20 nm shorter than the cross-over wavelength when the wavelength at the crossing point between the transmittance spectrum at a longer wavelength side of the excitation filter set and the transmittance spectrum at a shorter wavelength side of the absorption filter set is defined as a cross-over wavelength, thereby enabling to obtain a bright fluorescent image with a very high S/N ratio without requiring a filter with higher precision.

Claims

1. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in the detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set includes at least two interference films with a transmittance of 25% or more at a wavelength by 20 nm shorter than the cross-over wavelength, and satisfies the following condition:

space="preserve" listing-type="equation">0.05%<

T<

10%

where T denotes the transmittance of said interference film at said cross-over wavelength, and the cross-over wavelength is defined as a wavelength at the crossing point between the transmittance spectrum at a longer wavelength side of said excitation filter set and the transmittance spectrum at a shorter wavelength side of said absorption filter set.

13. A fluorescence detecting device according to any one of claims 1 to 4, wherein at least one of the interference films are disposed by being tilted against the plane perpendicular to the optical axis.

14. A fluorescence detecting device according to claim 13, wherein the interference film is formed on a transparent substrate adjustably mounted on the device.

15. A fluorescence detecting device according to any one of claims 1 to 12, wherein the interference film is disposed in non-parallel relations with each other.

16. A fluorescence detecting device according to any one of claims 1 to 4, wherein the interference film satisfies the condition of 0.1%<

T"<

5% when T" is defined to be the transmittance of the interference film at the cross-over wavelength or at the central wavelength of said light source.

2. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in the detecting optical system for allowing the fluorescence to transmit, wherein

said absorption filter set includes at least two interference films with a transmittance of 50% or more at a wavelength by 20 nm longer than the cross-over wavelength, and satisfies the following condition:

space="preserve" listing-type="equation">0.05%<

T<

10%

where T denotes the transmittance of said interference film at said cross-over wavelength, and the cross-over wavelength is defined as a wavelength at the crossing point between the transmittance spectrum at a longer wavelength side of said excitation filter set and the transmittance spectrum at a shorter wavelength side of said absorption filter set.

3. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in the detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set includes at least two first interference films with a transmittance of 25% or more at a wavelength by 20 nm shorter than the cross-over wavelength, said absorption filter set includes at least two second interference films with a transmittance of 50% or more at a wavelength by 20 nm longer than the cross-over wavelength, and said first and second interference films satisfy the following condition:

space="preserve" listing-type="equation">0.05%<

T<

10%

where T denotes the transmittance of said interference film at said cross-over wavelength, and the cross-over wavelength is defined as a wavelength at the crossing point between the transmittance spectrum at a longer wavelength side of said excitation filter set and the transmittance spectrum at a shorter wavelength side of said absorption filter set.

4. A fluorescence detecting device comprising a monochromatic or semi-monochromatic light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, a detecting optical system for detecting the fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

the shorter wavelength side of the fluorescence transmission wavelength band of said absorption filter set includes at least two interference films with a transmittance of 50% or more at a wavelength by 20 nm longer than the central wavelength of said light source, and satisfies the following condition:

space="preserve" listing-type="equation">0.05%<

T.sub.1 <

10%

where T₁ denotes the transmittance of said interference film at the central wavelength of said light source.

5. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set has a plurality of excitation light transmission wavelength bands τ

_i (i=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_i (i=1, 2, . . . , n) corresponding to the excitation light transmission wavelength bands τ

_i, said excitation filter set including at least two interference films with a transmittance of 25% or more at a wavelength by 20 nm shorter than the cross-over wavelength λ

_i and satisfying the following condition;

space="preserve" listing-type="equation">0.05%<

T.sub.i <

10%

where T_i denotes the transmittance of said interference film at said cross-over wavelength λ

_i, and the cross-over wavelength λ

_i is defined as a wavelength at the crossing point between the transmittance spectrum at a longer wavelength side of an arbitrary excitation light transmission wavelength band τ

_i and the transmittance spectrum at a shorter wavelength side of the fluorescence transmission wavelength band E_i.

6. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set has a plurality of excitation light transmission wavelength bands τ

_i (i=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_i (i=1, 2, . . . , n) corresponding to the excitation light transmission wavelength bands τ

_i, said absorption filter set including at least two interference films with a transmittance of 50% or more at a wavelength by 20 nm longer than the cross-over wavelength λ

_i and satisfying the following condition;

space="preserve" listing-type="equation">0.05%<

T.sub.i <

10%

where T_i denotes the transmittance of said interference film at said cross-over wavelength λ

_i, and the cross-over wavelength λ

_i is defined as a wavelength at the crossing point between the transmittance spectrum at a longer wavelength side of an arbitrary excitation light transmission wavelength band τ

_i and the transmittance spectrum at a shorter wavelength side of the fluorescence transmission wavelength band E_i.

7. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said the illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set has a plurality of excitation light transmission wavelength bands τ

_i (i=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_i (i=1, 2, . . . , n) corresponding to the excitation light transmission wavelength bands τ

_i, said excitation filter set including at least two first interference films with a transmittance of 25% or more at a wavelength by 20 nm shorter than the cross-over wavelength λ

_i, said absorption filter set including at least two second interference films with a transmittance of 50% or more at a wavelength by 20 nm longer than the cross-over wavelength λ

_i, and said first and second interference films satisfy the following condition;

space="preserve" listing-type="equation">0.05%<

T.sub.i <

10%

where T_i denotes the transmittance of said interference film at said cross-over wavelength λ

_i, and the cross-over wavelength λ

_i is defined as a wavelength at the crossing point between the transmittance spectrum at a longer wavelength side of an arbitrary excitation light transmission wavelength band τ

_i and the transmittance spectrum at a shorter wavelength side of the fluorescence transmission wavelength band E_i.

8. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set has a plurality of excitation light transmission wavelength bands τ

_i (i=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_i (i=1, 2, . . . , n) corresponding to the excitation light transmission wavelength bands τ

_i, said excitation filter set including at least two interference films with a transmittance of 25% or more at a wavelength by 20 nm longer than the cross-over wavelength λ

_i '"'"', and said interference films satisfy the following condition;

space="preserve" listing-type="equation">0.05%<

T'"'"'.sub.i <

10%

where T'"'"'_i denotes the transmittance of said interference film at said cross-over wavelength λ

'"'"'_i, and the cross-over wavelength λ

'"'"'_i is defined as a wavelength at the crossing point between the transmittance spectrum at a shorter wavelength side of an arbitrary excitation light transmission wavelength band τ

_i +1 and the transmittance spectrum at a longer wavelength side of the fluorescence transmission wavelength band E_i.

9. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set has a plurality of excitation light transmission wavelength bands τ

_i (i=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_i (i=1, 2, . . . , n) corresponding to the excitation light transmission wavelength bands τ

_i, said absorption filter set including at least two interference films with a transmittance of 50% or more at a wavelength by 20 nm shorter than the cross-over wavelength λ

_i '"'"', and said interference films satisfy the following condition;

space="preserve" listing-type="equation">0.05%<

T'"'"'.sub.i <

10%

where T'"'"'_i denotes the transmittance of said interference film at said cross-over wavelength λ

'"'"'_i, and the cross-over wavelength λ

'"'"'_i is defined as a wavelength at the crossing point between the transmittance spectrum at a shorter wavelength side of an arbitrary excitation light transmission wavelength band τ

_i +1 and the transmittance spectrum at a longer wavelength of the fluorescence transmission wavelength band E_i.

10. A fluorescence detecting device comprising a light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, an excitation filter set disposed in said illumination optical system for allowing a light with a specified wavelength region to transmit, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said excitation filter set has a plurality of excitation light transmission wavelength bands τ

_i (i=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_i (i=1, 2, . . . , n) corresponding to the excitation light transmission wavelength bands τ

_i, said excitation filter set including at least two first interference films with a transmittance of 25% or more at a wavelength by 20 nm longer than the cross-over wavelength λ

_i '"'"', said absorption filter set including at least two second interference films with a transmittance of 50% or more at a wavelength by 20 nm shorter than the cross-over wavelength λ

_i '"'"', and said first and second interference films satisfy the following condition;

space="preserve" listing-type="equation">0.05%<

T'"'"'.sub.i <

10%

where T'"'"'_i denotes the transmittance of said interference film at said cross-over wavelength λ

'"'"'_i, and the cross-over wavelength λ

'"'"'_i is defined as a wavelength at the crossing point between the transmittance spectrum at a shorter wavelength of an arbitrary excitation light transmission wavelength band τ

_i +1 and the transmittance spectrum at a longer wavelength of the fluorescence transmission wavelength band E_i.

11. A fluorescence detecting device comprising a monochromatic or semi-monochromatic light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said light source emits a light with a plurality of central wavelengths of λ

1_j (j=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_j (j=1, 2, . . . , n) corresponding to the central wavelength λ

1_j of said light source, the shorter wavelength side of the fluorescence transmission wavelength bands E_j of said absorption filter set including at least two interference films with a transmittance of 50% or more at a wavelength by 20 nm longer than the central wavelength λ

1_j of said light source and satisfying the following condition;

space="preserve" listing-type="equation">0.05%<

T1.sub.j <

10%

where T1_j denotes the transmittance of the interference film at the central wavelengths of said light source.

12. A fluorescence detecting device comprising a monochromatic or semi-monochromatic light source, an illumination optical system for irradiating the light emitted from said light source to a specimen, a detecting optical system for detecting a fluorescence emitted from said specimen, and an absorption filter set disposed in said detecting optical system for allowing the fluorescence to transmit, wherein

said light source emits a light with a plurality of central wavelengths of λ

1_j (j=1, 2, . . . , n), and said absorption filter set has a plurality of fluorescence transmission wavelength bands E_j (j=1, 2, . . . , n), corresponding to the central wavelength λ

1_j of said light source, the longer wavelength side of the fluorescence transmission wavelength bands E_j of said absorption filter set including at least two interference films with a transmittance of 50% or more at a wavelength by 20 nm shorter than the central wavelength λ

1_j +1 of said light source and satisfying the following condition;

space="preserve" listing-type="equation">0.05%<

T'"'"'1.sub.j <

10%

where T'"'"'1_j denotes the transmittance of the interference film at the central wavelength of said light source.