Laser excitation fluorescent microscope

• US 8,310,754 B2
• Filed: 03/26/2010
• Issued: 11/13/2012
• Est. Priority Date: 10/31/2007
• Status: Active Grant

• Current Assignee: Nikon Corporation
• Original Assignee: Nikon Corporation
• Inventors: Okugawa, Hisashi, Aikawa, Naoshi, Sato, Masatoshi
• Primary Examiner(s): Amari, Alessandro
• Application Number:
  US12/748,031
• Publication Number:
  US 20100182683A1
• Time in Patent Office: 963 Days
• Field of Search: 359/363, 359368-380, 359381-389, 356/318
• US Class Current: 359/385
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 5/0833 comprising inorganic materials only
• CPC Class Codes: G02B 5/285 comprising deposited thin solid films G02B5/281 - G02B5/289 take precedence; multilayered film filters for fibre optic multiplexing G02B6/29361

Abstract

The present application has a proposition to provide a highly efficient laser excitation fluorescent microscope. Accordingly, a laser excitation fluorescent microscope of the present application includes a laser light source part radiating at least two types of excitation lights having different wavelengths; a light collecting part collecting the two types of excitation lights on a sample; a high-functional dichroic mirror, disposed between the laser light source part and the light collecting part, reflecting the two types of excitation lights to make the excitation lights incident on the light collecting part, and transmitting two types of fluorescence generated at the sample; and a detecting part detecting light transmitted through the high-functional dichroic mirror, in which an incident angle θ of the excitation lights and the fluorescence to the high-functional dichroic mirror satisfies a formula of 0°<θ<45°.

Claims

1. A laser excitation fluorescent microscope, comprising:

a light collecting part collecting at least two types of excitation lights radiated by laser light source part on a sample;

a dichroic mirror, disposed between the laser light source part and the light collecting part, making the two types of excitation lights incident on the light collecting part by reflecting the two types of excitation lights, and transmitting two types of fluorescence generated at the sample in accordance with the two types of excitation lights; and

a detecting part detecting each of the two types of fluorescence transmitted through the dichroic mirror, in which the detecting part includes a plurality of detectors, wherein an incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror is smaller than 25°

, and a transmittance of each of the two types of fluorescence to the dichroic mirror is 95% or more.

2. The laser excitation fluorescent microscope according to claim 1, wherein

the incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror satisfies a formula of 10°

<

θ

<

25°

.

3. The laser excitation fluorescent microscope according to claim 1, wherein

the incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror satisfies a formula of 10°

<

θ

<

15°

.

4. The laser excitation fluorescent microscope according to claim 1, wherein

the incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror is 12°

.

5. The laser excitation fluorescent microscope according to claim 1, wherein

a separating layer of the dichroic mirror is formed by a dielectric multilayer.

6. The laser excitation fluorescent microscope according to claim 1, wherein the dichroic mirror includes a separating layer formed by a dielectric multilayer in which a film thickness of each dielectric film forming the dielectric multilayer is set to a non-integral multiple of λ

/4.

7. A laser excitation fluorescent microscope, comprising:

a light collecting part collecting at least two types of excitation lights radiated by a laser light source part on a sample;

a dichroic mirror, disposed between the laser light source part and the light collecting part, making the two types of excitation lights incident on the light collecting part by reflecting the excitation lights, and transmitting two types of fluorescence generated at the sample in accordance with the two types of excitation lights, the dichroic mirror being formed by a dielectric multilayer; and

a detecting part detecting each of the two types of fluorescence transmitted through the dichroic mirror, in which the detecting part includes a plurality of detectors, wherein a wavelength characteristic curve of a reflectivity and a transmittance of the dichroic mirror has;

a first reflecting band covering a wavelength band of one of the two types of excitation lights;

a first transmitting band covering a wavelength band of one of the fluorescence generated in accordance with said one of the two types of excitation lights;

a second reflecting band covering a wavelength band of other one of the two types of excitation lights; and

a second transmitting band covering a wavelength band of one of the fluorescence generated in accordance with said other one of the two types of excitation lights, wherein;

a reflectivity in each of the first reflecting band and the second reflecting band is 95% or more;

a transmittance in each of the first transmitting band and the second transmitting band is 95% or more;

a wavelength width T₁ of the first transmitting band and a wavelength width T₂ of the second transmitting band are each 25 nm or more; and

a rising width A₁ from the first reflecting band to the first transmitting band and a rising width A₂ from the second reflecting band to the second transmitting band are each 6 nm or less.

8. The laser excitation fluorescent microscope according to claim 7, wherein

the transmittance in each of the first transmitting band and the second transmitting band indicates a value of 98% or more for a range of 90% or more of the wavelength width.

9. The laser excitation fluorescent microscope according to claim 7, wherein

a gap B between the first transmitting band and the second transmitting band is 20 nm or less.

10. The laser excitation fluorescent microscope according to claim 7, wherein

an incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror satisfies a formula of 0°

<

θ

<

45°

.

11. The laser excitation fluorescent microscope according to claim 7, wherein

the incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror satisfies a formula of 10°

<

θ

<

25°

.

12. The laser excitation fluorescent microscope according to claim 7, wherein

the incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror satisfies a formula of 10°

<

θ

<

15°

.

13. The laser excitation fluorescent microscope according to claim 7, wherein

the incident angle θ

of the excitation lights and the fluorescence to the dichroic mirror is 12°

.

14. The laser excitation fluorescent microscope according to claim 7, wherein

the detecting part detects a spectrum of light transmitted through the dichroic mirror.