System and method for increasing the contrast of an image produced by an epifluorescence microscope

• US 20020141050A1
• Filed: 03/19/2002
• Published: 10/03/2002
• Est. Priority Date: 03/19/2001
• Status: Active Grant

• Current Assignee: Ikonisys, Inc.
• Original Assignee: Ikonisys, Inc.
• Inventors: Tafas, Triantafyllos, Tsipouras, Petros
• Granted Patent: US 6,956,695 B2
• Time in Patent Office: Days
• US Class Current: 359/385
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/082 for incident illumination only
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/34 Microscope slides, e.g. mounting specimens on microscope slides

Abstract

The contrast of an image produced by epifluorescence microscopy may be increased by placing a high-pass dichroic reflecting film behind the sample. The reflecting film reflects the emission light emitted by the fluorescent tags in the sample back through the objective lens while allowing the shorter wavelength excitation light to pass through the sample holder.

Claims

1. An epifluorescence microscope for imaging a biological sample having fluorescent tag molecules, the tag molecules emitting an emission light at an emission frequency when illuminated by an excitation light having an excitation frequency, the microscope comprising:

an excitation light source generating an excitation light;

a first dichroic mirror reflecting the excitation light;

an objective lens disposed to receive the excitation light reflected by the dichroic mirror and to illuminate the sample with the excitation light;

an imaging lens disposed to receive emission light from the sample through the objective lens and first dichroic mirror; and

a dichroic sample reflector disposed behind the sample reflecting the emission light back through the sample, objective lens, first dichroic mirror and imaging lens, while transmitting the excitation light through the reflector.

2. A sample holder for supporting a sample for epifluorescence microscopy, the sample emitting an emission light when illuminated by an excitation light, the sample holder comprising:

a base; and

a dichroic reflector disposed on the base, wherein the dichroic reflector reflects the emission light emitted by the sample while transmitting the excitation light illuminating the sample.

3. A sample holder of claim 2 wherein the dichroic reflector is concave having a focal point disposed within the sample.

13. The sample holder of claim 2 wherein the base is a microscope slide.

4. A microscope slide for supporting a sample, the slide comprising:

a top surface; and

an infra-red reflecting film deposited on the top surface, the film directly supporting the sample.

5. The microscope slide of claim 4 wherein the infra-red reflecting film comprises titanium.

6. The microscope slide of claim 4 wherein the infra-red reflecting film has a thickness of 0.6 nm to 90 nm.

7. The microscope slide of claim 4 wherein the infra-red reflecting film reflects between 4 to 8% of an incident infra-red beam.

8. The microscope slide of claim 7 wherein the infra-red reflecting film reflects between 5.5 to 7% of the incident infra-red beam.

9. The microscope slide of claim 4 further comprising a bottom surface and a dichroic reflecting film disposed on the bottom surface of the microscope slide.

10. The microscope slide of claim 9 wherein the dichroic reflecting film reflects an emission light emitted by the sample when illuminated by an excitation light, the dichroic reflecting film absorbing the excitation light.

11. The microscope slide of claim 9 wherein the dichroic reflecting film reflects an emission light emitted by the sample when illuminated by an excitation light, the dichroic reflecting film transmitting the excitation light.

12. A method for increasing the contrast of an image produced by an epifluorescence microscope of a sample emitting an emission light when illuminated by an excitation light comprising the steps of:

illuminating the sample with the excitation light;

collecting a first portion of the emission light;

reflecting a second portion of the emission light;

collecting the reflected portion of the emission light; and

producing an image using the collected first portion of the emission light and the collected reflected portion of the emission light.