Patent Publication Number: US-2009236543-A1

Title: Fluorescence Detection Using Lyman-alpha Line Illumination

Description:
RELATED APPLICATION/CLAIM OF PRIORITY 
     This application is related to and claims priority from provisional application Ser. No. 61/038,025, filed Mar. 19, 2008, which provisional application is incorporated by reference herein. 
    
    
     BACKGROUND 
     The Hydrogen Lyman-α radiation line light at the wavelength of 121.6 nm is normally considered to be within the VUV (vacuum ultra-violet) band. However, the present invention is based on the recognition that this wavelength is particularly convenient for optical applications because it has substantial atmospheric transmission. 
     SUMMARY OF THE PRESENT INVENTION 
     The present invention takes advantage of the atmospheric transmission properties of the Hydrogen Lyman-α radiation line (121.6 nm wavelength) to illuminate a sample with high energy VUV photons at least partially in an atmospheric environment (without the need for a vacuum environment). The high energy illuminating photons generate luminescent radiation from the sample at longer wavelengths, typically in the visible wavelength range, and this radiation can then be imaged, e.g. with a normal visible microscope. 
     Other features of the present invention will be apparent from the following detailed description and the accompanying drawings 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIGS. 1   a  and  1   b  schematically illustrate two exemplary ways to illuminating a sample with high energy UV photons in an atmospheric environment, in accordance with the principles of the present invention. 
     
    
    
     DETAILED DESCRIPTION 
     As described above, the present invention takes advantage of the atmospheric transmission properties of the Hydrogen Lyman-α radiation line (121.6 nm wavelength) to illuminate a sample with high energy VUV photons in an atmospheric environment (without the need for a vacuum environment). The high energy illuminating photons generate luminescent radiation from the sample at longer wavelengths, typically in the visible wavelength range, and this radiation can then be imaged with a normal visible microscope. 
       FIGS. 1   a  and  1   b  schematically illustrate three illumination conditions that apply the illumination principles of the present invention. In each of the figures, a source  100  generates light at the Hydrogen Lyman-α radiation line (121.6 nm wavelength), and that light is directed at a sample  102 . Luminescent radiation from the sample  102  is then detected by a detector  104  which can be, e.g., part of a visible microscope. 
     In each of the figures, the source  100  comprises a lamp  100   a  or similar device that produces light at the Hydrogen Lyman-α radiation line (121.6 nm wavelength) and a concave reflector  100   b,  which reflects the Lyman-α radiation that is directed at the sample. Preferably, the source (i.e. lamp  100   a  and concave mirror  100   b  in  FIG. 1   a,  and lamp  100   a  and optical components of a catadioptric optical system described further below) may be disposed in an atmosphere that is substantially free of oxygen, so that the oxygen does not interfere with the desired transmission of Lyman-α radiation at the sample  102 . Moreover, in all of the disclosed embodiments, Lyman-α radiation from the source  100  is directed at the sample at least partially in an atmospheric environment, as further described below. 
       FIG. 1   a  illustrates two illumination conditions for illuminating the sample  102  with light at the Hydrogen Lyman-α radiation line (121.6 nm wavelength). In one illumination condition, light from the source  100  illuminates the sample  102  with Lyman-α radiation reflected from concave mirror  100   b  from the mirror orientation labeled A. The illumination of the sample from that orientation is sometimes referred to as “bright field” illumination, because light from the source at the Lyman-α radiation line is from an orientation that is substantially in line with the detector  104  that is part of the microscope that detects luminescent radiation from the sample. Moreover, in accordance with the principles of the present invention, at least a portion of the transmission of Lyman-α radiation is in an atmospheric environment (i.e. not in a vacuum environment). Thus, in the “bright line” illumination condition of  FIG. 1   a,  the sample  102  is located in an environment that is completely exposed to atmosphere, and transmission of Lyman-α radiation directed from the source  100  at the sample is at least partially through that atmospheric environment. Thus, the transmission takes advantage of the ability to transmit Lyman-α radiation in an atmospheric environment, and by locating the sample in that atmospheric environment, the sample can be easily changed, without having to enter a chamber or other enclosure that controls the atmosphere in which the sample is located. 
     In another illumination condition illustrated in  FIG. 1   a,  light from the source  100  illuminates the sample  102  with Lyman-α radiation reflected from concave mirror  100   b  from the mirror orientation labeled B. The illumination of the sample from that orientation is sometimes referred to as “dark field” illumination, because light from the source at the Lyman-α radiation line is from an orientation that is substantially oblique with respect to the cone of light directed into the optical system to the detector  104  that is part of the microscope that detects luminescent radiation from the sample. Thus, in the “dark field” illumination condition of  FIG. 1   a,  the sample  102  is also located in an environment that is completely exposed to atmosphere, and transmission of Lyman-α radiation directed from the source  100  at the sample is at least partially through that atmospheric environment. 
     Accordingly, in each of the illumination conditions shown in  FIG. 1   a,  the illumination of the sample  102 , at the Lyman-α radiation line is at least partly in an atmospheric environment. Thus, the transmission takes advantage of the ability to transmit Lyman-α radiation in an atmospheric environment and by locating the sample in that atmospheric environment, the sample can be easily changed, without having to enter a chamber or other enclosure that controls the atmosphere in which the sample is located. 
       FIG. 1   b  illustrates a “bright field” environmental configuration where catadioptic imaging optics effectively form part of the source  100 , and are shared by the illumination system, so that “bright field” illumination of the sample  102  is provided, at Lyman-α radiation line, at least partly in an atmospheric environment, and luminescent radiation from the sample  102  is detected by the detector  104  which can comprise, e.g. a part of a visible microscope. In the illumination system and method of  FIG. 1   b,  the source of light at the Lyman-α radiation line is produced by a source that includes the lamp  100   a,  concave mirror  100   b,  and catadioptric optics comprising a beam splitter  106 , a convex reflector  108 , and one of a pair of concave mirrors  110 . Luminescent radiation from the sample is reflected from one of the concave mirrors  110 , the convex reflector  108  and is directed through beam splitter  106  and to the detector  104 . The sample  102  is located in an atmospheric environment that encompasses at least part of the optical path between the mirrors  110  and the sample  102 . 
     In all of the illustrated embodiments, the path of the Lyman-α radiation is shown with dashed line. 
     Although  FIGS. 1   a  and  1   b  generally show the Lyman-α radiation directed with reflective optics, there are optical materials that could be used for transmissive elements. LiF and MgF 2  both have significant transmission at this wavelength, at least for thin elements like the beam splitter  106  shown in  FIG. 1   b,  and possibly for small lens elements near the sample. Thus, while the “source”  100  shown in the figures comprises the lamp  100   a  and the concave mirror  100   b,  the source could also include a lamp and a transmissive element. 
     Although other applications of the Lyman-α line are known, and although fluorescence microscopy is also well known, the use of Lyman-α radiation for illumination in fluorescence microscopy, at least partially in an atmospheric environment, and according to the principles of the present invention, is new. 
     An advantage of this invention is that using illumination with such a short wavelength (121.6 nm) should expand the range of fluorophores that can be excited and imaged. This is conveniently enabled by the choice of wavelength, since the radiation can be readily generated with a Hydrogen Lyman-α source, and since this atmosphere is relatively transmissive at this wavelength. 
     Furthermore, since the imaging optics do not have to transmit the illuminating radiation, this invention could be embodied as an attachment to an existing visible microscope, provided that the fluorescent wavelength is within the transmission bandwidth of the optics. For example, the principles of the present invention can be used with a microscope such as shown in U.S. Pat. No. 6,337,767, which is assigned to the assignee of the present invention, and incorporated herein by reference. The microscope disclosed in that patent is configured to detect both radiation in the visible range, and also radiation in the ultraviolet range. Thus, if luminescence from the sample, produced according to the principles of the present invention, is in the visible range, that luminescence can be detected by the microscope in its visible detection mode. On the other hand, if luminescence from the sample is in the ultraviolet range (especially the near ultraviolet range), that luminescence can also be detected by the microscope in its ultraviolet mode. 
     Accordingly, the foregoing description illustrates and describes how the principles of the present invention provide for illuminating a sample by radiation at the Hydrogen Lyman-α radiation line (121.6 nm wavelength), at least partially in an atmospheric environment, and detecting luminescent radiation from the sample at longer wavelengths. 
     With the foregoing description in mind, the manner in which the principles of the present invention can be used to provide various systems and methods for illuminating a sample using the Hydrogen Lyman-α radiation line (121.6 nm wavelength) in an atmospheric environment will be apparent to those in the art.