Fluorescence microscope and observation method using the same

• US 20050092934A1
• Filed: 10/28/2004
• Published: 05/05/2005
• Est. Priority Date: 10/29/2003
• Status: Active Grant

• Current Assignee: Korea Electrotechnology Research Institute
• Original Assignee: Korea Electrotechnology Research Institute
• Inventors: Papayan, Garry V., Kang, Uk
• Granted Patent: US 7,297,961 B2
• Time in Patent Office: Days
• US Class Current: 250/458.1
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

The present invention relates, in general, to a fluorescence microscope and method of observing samples using the microscope and, more particularly, to a fluorescence microscope and method of observing samples using the microscope, which can reduce optical noise and obtain images with higher sensitivity, thus obtaining precise information about the density, quantity, location, etc. of a fluorophore, and which can simultaneously process separate images even when a plurality of fluorophores having different excitation and fluorescent wavelength ranges is distributed, thus easily obtaining information about the fluorophores. The fluorescence microscope of the present invention includes an objective lens, and first and third medium units. The first medium unit has a refractive index of n₁ to accommodate one or more micro-objects including fluorophores and provide a path of excitation light to excite the fluorophores. The third medium unit has a refractive index of n₃, and is placed between the first medium unit and the objective lens to totally reflect the excitation light incident through the first medium unit at an interface of the third medium unit coming into contact with the first medium unit. The refractive indices of the third and first medium units satisfy a relationship of n₁>n₃.

Claims

1. A fluorescence microscope, comprising:

an objective lens;

a first medium unit with a refractive index of n₁ for accommodating one or more micro-objects including fluorophores and providing a path of excitation light to excite the fluorophores; and

a third medium unit with a refractive index of n₃ placed between the first medium unit and the objective lens to totally reflect the excitation light incident through the first medium unit at an interface of the third medium unit coming into contact with the first medium unit,

wherein the refractive indices of the third and first medium units satisfy a relationship of n₁>

n₃,

wherein an incident angle of the excitation light on the interface is equal to or greater than a critical angle, and

wherein fluorescence, emitted when fluorophores present in micro-objects that exist in the path of the excitation light within the first medium unit are excited, is captured by the objective lens, thus observing the micro-objects.

4. The fluorescence microscope according to any of claims 1 to 3, further comprising:

an image processing unit for providing images of objects obtained due to fluorescence collected by the objective lens as images;

a prism including at least an incident surface, a contact surface and an output surface;

a light source for providing excitation light to the incident surface of the prism; and

an optical coupling unit formed on the contact surface of the prism so as to allow a sample including one or more micro-objects having fluorophores to be seated thereon.

5. The fluorescence microscope according to claim 4, further comprising an absorber installed on the output surface of the prism to absorb the excitation light that has been totally reflected.

6. The fluorescence microscope according to claim 4, wherein the optical coupling unit is made of an elastic material to facilitate optical contact with the seated sample.

2. A fluorescence microscope, comprising:

an objective lens;

a first medium unit with a refractive index of n₁ for accommodating one or more micro-objects including fluorophores and providing a path of excitation light to excite the fluorophores;

a fourth medium unit with a refractive index of n₄ for covering a surface facing the objective lens among surfaces of the first medium unit; and

a third medium unit with a refractive index of n₃ placed between the fourth medium unit and the objective lens to totally reflect the excitation light incident through the first and fourth medium units at an interface of the third medium unit coming into contact with the fourth medium unit,

wherein the refractive indices of the first, third and fourth medium units satisfy a relationship of n₁≈

n₄>

n₃,

wherein an incident angle of the excitation light on the interface between the fourth and third medium units is equal to or greater than a critical angle, and

wherein fluorescence, emitted when fluorophores present in micro-objects that exist in the path of the excitation light within the first medium unit are excited, is captured by the objective lens, thus observing the micro-objects.

3. The fluorescence microscope according to claim 2, further comprising an optical coupling fluid layer for optically coupling the fourth and first medium units to each other as a fifth medium unit, the fourth medium unit being a cover glass,

wherein the fifth medium unit has a refractive index of n₅, and satisfies a relationship of n₅≈

n₄≈

n₁.

7. An observation method using a fluorescence microscope, comprising the steps of:

allowing excitation light, required to excite fluorophores, to pass through a medium accommodating one or more micro-objects including fluorophores;

totally reflecting the excitation light to prevent the excitation light from reaching an objective lens; and

allowing the totally reflected excitation light to pass again through the medium accommodating one or more micro-objects,

wherein fluorescence, emitted when fluorophores present in micro-objects that exist in the path of the excitation light within the first medium unit are excited, is captured by the objective lens, thus observing the micro-objects.