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  System and method for increasing the contrast of an image produced by an epifluorescence microscopeUSPTO Application #: 20080100911Title: System and method for increasing the contrast of an image produced by an epifluorescence microscope Abstract: The contrast of an image produced by epifluorescence microscopy may be increased by placing a dichroic reflector behind the sample. The dichroic reflector 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. (end of abstract) Agent: Kelley Drye & Warren LLP - Stamford, CT, US Inventors: Triantafyllos Tafas, Petros Tsipouras USPTO Applicaton #: 20080100911 - Class: 359385000 (USPTO) The Patent Description & Claims data below is from USPTO Patent Application 20080100911. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This is a divisional application of continuation application U.S. patent application Ser. No. 11/225,460, filed Sep. 13, 2005, and U.S. patent application Ser. No. 10/102,500, filed Mar. 19, 2002, which claims priority of U.S. Provisional Application No. 60/276,906, filed Mar. 19, 2001; the entire contents of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION [0002] The present invention relates to microscope slides and the like for use in epifluorescence microscopy of biological specimens. BACKGROUND OF THE INVENTION [0003] Citation or identification of any reference in this section or any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention. [0004] An epifluorescence microscope is similar to a conventional reflecting optical microscope in that both microscopes illuminate the sample and produce a magnified image of the sample. An epifluorescence microscope, however, uses the emitted fluorescent light to form an image whereas a conventional reflecting optical microscope uses the scattered illumination light to form an image. The epifluorescent microscope uses a higher intensity illumination, or excitation, light than a conventional microscope. The higher intensity excitation light is needed to excite a fluorescent molecule in the sample thereby causing the fluorescent molecule to emit fluorescent light. The excitation light has a higher energy, or shorter wavelength, than the emitted light. The epifluorescence microscope uses the emitted light to produce a magnified image of the sample. The advantage of a epifluorescence microscope is that the sample may be prepared such that the fluorescent molecules are preferentially attached to the biological structures of interest thereby producing an image of the biological structures of interest. [0005] A common problem in epifluorescence microscopy is the low contrast, or low signal-to-noise (S/N) ratio, of the fluorescent image. This is due to the low intensity of the emitted light compared to the high intensity of the excitation light. A dichroic mirror is usually used to reduce the scattered excitation light before the image is viewed or recorded. [0006] The dichroic mirror is only partially effective in removing the excitation light from the emitted light so other measures must be taken to increase the S/N ratio of the fluorescent image. In order to assist in the discussion of the other approaches to increasing the S/N ratio of the fluorescent image, reference to FIG. 1 is helpful. [0007] FIG. I illustrates the optical path and components of a typical epifluorescence microscope. A sample 100 is placed on a sample holder 105, which is normally a microscope slide. The sample is prepared prior to being placed on the holder 105 with fluorescent tags that bind to the biological structures of interest. The fluorescent tags may be a single type of fluorescent tag that binds to a particular biological structure or may be a mixture of several fluorescent tag types with each tag type binding to a different biological structure. The sample 100 is illuminated by a light source 110 that produces the excitation light with sufficient intensity to cause the tags to emit fluorescent light. The excitation light generated by the light source 110 follows a path 115 through an excitation filter 120 that acts as a band-pass filter allowing only a narrow range of frequencies to pass through the excitation filter 120. The excitation filter 120 is chosen to allow only the light of a frequency that will cause the tags to fluoresce. The excitation light is reflected by a dichroic mirror 130 into the objective lens 140 of the microscope following path 125. A dichroic mirror separates the excitation light from the emission light, in this example, by reflecting the excitation light while transmitting the emission light. The excitation light propagates through the objective lens 140 and illuminates the sample 100 and excites the tags in the sample to emit fluorescent light, also referred to as emission light. The emission light propagates along path 125 in the opposite direction as the excitation light. The emission light passes through the objective lens 140 and through the dichroic mirror 130 and continues along path 135 through an emission filter 150. The emission filter 150 is selected to allow only light matching the frequency of the emission light to pass through the filter. The emission filter 150 may be a band-pass fitter, or a long-pass filter that allows the longer wavelength emission light to pass through while stopping the shorter wavelength excitation light. After filtering by the emission filter 150, the emission light is formed into an image by an imaging lens 160. If the emission filter 150 is perfectly efficient in removing all but the emission light, the magnified fluorescent image would have a very high contrast and S/N ratio. Unfortunately, emission filters are not perfectly efficient so a small amount of excitation light is transmitted though the emission filters. Because the intensity of the excitation light is very high, the small fraction of excitation light that passes through the emission filter is sufficient to severely degrade the contrast of the fluorescent image. In addition, the excitation frequency is usually very close to the emission frequency of the fluorescent tag molecule. The closeness of the two frequencies adds a further requirement on the emission filter that the filter have a very steep adsorption edge between the emission frequency and excitation frequency. [0008] U.S. Pat. No. 6,094,274 issued on Jul. 25, 2000 to Yokoi teaches the use of two interference films as an emission filter. The two interference films act to sharpen the adsorption edge between the emission frequency and excitation frequency. The sharp adsorption edge blocks more of the excitation light while transmitting more of the emission light to the imaging lens. [0009] Another approach to increasing the S/N ratio of a fluorescent image is disclosed in Japanese Application Publication No. 9-292572 by Sudo, et al. published on Nov. 11, 1997 (hereinafter referred to as "Sudo"). Sudo discloses the use of a mirror behind the sample that reflects the excitation light back through the sample. The reflected excitation light approximately doubles the excitation light seen by the sample and therefore approximately doubles the amount of emission light given off by the sample. A portion of the reflected excitation light will, however, also pass through the dichroic mirror and emission filter adding to the "noise" of the higher emission signal. In addition, the increased illumination of the sample from the reflected excitation light increases the bleaching effect on the tagged sample. Bleaching occurs when the fluorescent tag molecules emit decreasing amounts of fluorescent light as the molecules are illuminated by the excitation light. For example, a fluorescent tag molecule will emit less than 10% of its emission intensity after only a minute of being illuminated by the excitation light. As the intensity of the excitation light increases the bleaching rate increases thereby decreasing the emission light and reducing the contrast of the fluorescent image. [0010] Therefore, there still remains a need to provide a microscope system capable of producing a high contrast fluorescent image while reducing unnecessary bleaching of the sample. SUMMARY OF THE INVENTION [0011] One aspect of the present invention is directed to 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. [0012] Another aspect of the present invention is directed to 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. [0013] Another aspect of the present invention is directed to 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. [0014] Another aspect of the present invention is directed to a sample bolder holding a sample for an epifluorescence microscope, the sample emitting an emission light when illuminated by an excitation light, the sample holder comprising: a base supporting the sample; and a sample reflector disposed on the base between the sample and base, wherein the reflector reflects the emission light emitted by the sample while transmitting the excitation light illuminating the sample, wherein the sample reflector is concave having a focal point disposed in the sample. Another aspect of the present invention is directed to a sample holder for supporting a sample emitting an emission light when illuminated by an excitation light, the sample holder comprising: a top surface for directly supporting a sample, the top surface having an infra-red reflecting film deposited on the top surface; and a bottom surface having a dichroic film deposited on the bottom surface, the dichroic film reflecting emission light and transmitting excitation light. [0015] Another aspect of the present invention is directed to a sample holder for supporting a sample emitting an emission light when illuminated by an excitation light, the sample holder comprising: a top surface for directly supporting a sample; and a dichroic film deposited on the top surface, the dichroic film transmitting excitation light and reflecting emission light. [0016] Another aspect of the present invention is directed to 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. BRIEF DESCRIPTION OF THE DRAWINGS [0017] The present invention may be understood more fully by reference to the following detailed description of the preferred embodiments of the present invention, illustrative examples of specific embodiments of the invention and the appended figures in which: [0018] FIG. 1 is a view of a conventional epifluorescence microscope. [0019] FIG. 2 is a view of an embodiment of the present invention. Continue reading... Full patent description for System and method for increasing the contrast of an image produced by an epifluorescence microscope Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this System and method for increasing the contrast of an image produced by an epifluorescence microscope patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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