Abstract:
The invention provides a fluorescence illumination adapter capable of being fitted to a wide range of existing stereomicroscopes. The fluorescence illumination adapter attaches to the stereomicroscope barrel that contains the imaging optics, and has high intensity light-emitting diodes that stimulate fluorescence in a specimen. A removable barrier filter is positioned in the optical path underneath the stereomicroscope barrel to prevent the fluorescence stimulating light entering the imaging optics. The light-emitting diodes are mounted on pivoting elements so that the location of the illumination spot on the specimen can be easily adjusted. The adapter also incorporates a white light-emitting diode source that can be used either for white-light viewing or for selective mixing with the fluorescence excitation illumination. The barrier filter is easily removed to facilitate rapid switching between fluorescence and white-light viewing.

Description:
FEDERALLY SPONSORED RESEARCH  
       [0001]     The United States Government may have rights in this invention under Contract DG133R-04-CN-0152 between the Department of Commerce (National Oceanic and Atmospheric Administration) and Physical Sciences Inc. 
     
    
     FIELD OF THE INVENTION  
       [0002]     This invention relates to systems and methods for providing an illumination adapter for stereomicroscopes, and more specifically to a removable adapter that converts a standard, visible light stereomicroscope into an external illumination, fluorescence stereomicroscope.  
       BACKGROUND OF THE INVENTION  
       [0003]     Many scientific, educational, and industrial disciplines benefit from fluorescence microscopy techniques for enhanced visualization.  
         [0004]     The fluorescence to be viewed may result from a number of sources, including but not limited to: naturally occurring fluorophores in the samples under investigation; molecular fluorescent tags that bind to features of interest; proteins of the green fluorescent protein family that are used to trace expression of genes; and dyes such as fluorescein that are injected to trace blood flow in the retina or other portions of the body.  
         [0005]     Because of their relatively low cost and great utility stereomicroscopes, many of them quite old, are found in many research and teaching laboratories. Researchers who want to add a fluorescence capability to their existing stereomicroscope currently have their options limited to making an ad hoc solution from individual components, purchasing a new stereomicroscope equipped with fluorescence capability, or purchasing a fluorescence adapter for an existing stereomicroscope.  
         [0006]     The last option is, however, limited because adapters are only available for specific makes or models of stereomicroscopes, mostly of recent vintage. Microscope manufacturers typically only offer adapters for their own lines, and third-party manufacturers offer adapter models tailored to specific models of stereomicroscopes. Furthermore, when fluorescence is being used to view subjects any non-fluorescing subjects in the field of view will not be clearly visible. It can be useful to mix a controlled amount of white light illumination with the fluorescence excitation to achieve a mixed reflected-light and fluorescence image, and this capability is not provided with existing adapters.  
         [0007]     Most systems for adapting a stereomicroscope for fluorescence provide a means to channel the excitation light into the objectives to achieve what is called epifluorescence illumination. This involves inserting an optical element between the eyepieces and the objectives, which requires engineering adapters that are specific to the stereomicroscope to be converted. For instance, U.S. Pat. No. 6,147,800 granted to Faber on Nov. 14, 2000 entitled “Fluorescence stereo microscope illuminator”, the contents of which are hereby incorporated by reference, is assigned to Kramer Scientific Corporation and describes a fluorescence adapter for stereomicroscopes that channels excitation light into both objectives. Adapters of this design are available from Kramer Scientific for specific stereomicroscope models. These adapters do not include the capability to mix white light and fluorescence excitation light.  
         [0008]     There are known in the art other approaches to implementing fluorescence capability for stereomicroscopes. Some of these are provided by stereomicroscope manufacturers for specific models and are integrated into the stereomicroscope. An example is the Olympus SZX-RFL3 fluorescence adapter for the Olympus Model SZX12 stereomicroscope, both supplied by Olympus Europa GmbH of Hamburg, Germany. This fluorescence adapter is not available for stereomicroscopes of other manufacturers, and can not be retrofitted to older instruments.  
         [0009]     Fluorescence adapters that provide the excitation light via a light source mounted on a stand next to the stereomicroscope stage are also known. Any illuminator mounted on a stand separate from the microscope has the disadvantage that it takes up valuable laboratory counter space, and can easily be knocked out of position by the operator while using the microscope controls or otherwise working in the vicinity of the microscope. Furthermore, these do not provide a convenient means of positioning the needed barrier filter in the emitted light path.  
         [0010]     The value of universal adapters for fluorescence is recognized in, for instance, U.S. Pat. No 5,349,468 issued to Rathbon et al. on Sep. 20, 1994 entitled “Adapter for microscope”, the contents of which are hereby incorporated by reference. This patent does not apply to stereomicroscopes, but describes a universal approach for adapting conventional compound microscopes for fluorescence viewing. The approach described in this patent is only suitable for microscopes having a single operational objective and could not be used with stereomicroscopes.  
         [0011]     Fluorescence illumination adapters for stereomicroscopes that provide epifluorescence illumination through the objectives are available only for limited, specific models of stereomicroscope. General-purpose solutions provided by illumination sources on stands external to the stereomicroscope take up bench space and are easily knocked out of alignment. Furthermore, these provide no convenient way of adding a barrier filter to the optical path. None of the available adapter systems provides a means of selectively mixing white-light illumination with the fluorescence excitation to better clarify the context in which the fluorescence is found.  
       SUMMARY OF THE INVENTION  
       [0012]     Briefly described, the invention relates to a system, method and apparatus for adapting an existing stereomicroscope for fluorescence microscopy. The adapter of this invention has several advantages, including being readily fitted to most existing stereomicroscopes, of being compact and unobtrusive, and of facilitating viewing by fluorescence and white light illumination simultaneously.  
         [0013]     In a preferred embodiment, these advantages may be realized in the following manner.  
         [0014]     The main body of the adapter may have a central hole that is large enough to fit over the largest, common stereomicroscope barrels. The body may be adapted to fit smaller diameters by various means such as thumbscrews, spring-loaded screws, or inserts of smaller inside diameter. The main body supports at least one, but preferably two or more, high intensity Light Emitting Diode (LED) light sources capable of exciting fluorescence. These light sources capable of exciting fluorescence may be pivot-mounted so as to be adjustably, rotatably attached to the main body so able to provide optimal illumination for fluorescence viewing at all viewing magnifications of the stereomicroscope. The fluorescence light sources may also be pivot out of the way if desired.  
         [0015]     In addition to the light sources capable of exciting fluorescence, the main body supports a pivot-mounted white LED that provides white-light illumination and is adjustably, rotatably attached to the main body. This enables the user to either mix white light in with the fluorescence excitation or easily switch between fluorescence and white-light viewing modes without removing the entire adapter. In a preferred embodiment, the emission barrier filter may be a material such as glass or plastic, having suitable optical and mechanical properties, that is attached magnetically underneath the hole in the main body so as to be easily removed. Small magnets, for instance, may be inset into the underside of the main body, and strips of metal that can be held by those magnets are attached to the upper surface of the barrier filter. The strength of the magnets is chosen so that the filter is held securely during use but may be easily removed.  
         [0016]     An additional piece of emission barrier filter is mounted to the back surface of the illuminator that will be in the front of the stereomicroscope when the adapter is installed. This acts as a light shield so that the user does not look directly at the bright excitation illumination spot on the stereomicroscope sample stage.  
         [0017]     The LEDs are controlled by an external unit containing the switches and circuitry needed to energize the white or fluorescence excitation LEDs separately or together, and a means to control the intensity of the white light.  
         [0018]     These and other features of the invention will be more fully understood by references to the following drawings. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0019]      FIG. 1  is a perspective view of the adapter mounted to a typical stereomicroscope.  
         [0020]      FIG. 2  is a perspective view of the adapter alone.  
         [0021]      FIG. 3  is graph showing the transmission spectrum of a yellow emission barrier filter.  
         [0022]      FIG. 4  is a bottom elevational view of the adapter showing the inset magnets for holding the emission barrier filter.  
         [0023]      FIG. 5  is a top elevational view of the barrier filter showing the attached metallic strips.  
         [0024]      FIG. 6  is a side cross-sectional view of one of the illuminator elements.  
         [0025]      FIG. 7  is an elevational view of one of the illuminator assemblies in its mounting bracket on the adapter.  
         [0026]      FIG. 8  is a schematic drawing showing the adapter connected to a control box and power supply. 
     
    
     DETAILED DESCRIPTION  
       [0027]     The present invention relates to fluorescence illumination adapters that enable a wide range of stereomicroscopes to be used for fluorescence imaging. In a preferred embodiment the fluorescence illumination adapter can accommodate a variety of stereomicroscope barrel diameters and allows simultaneous fluorescence excitation and white light illumination of a specimen using high intensity light-emitting diodes (LEDs).  
         [0028]     The potential to use high intensity LEDs instead of the arc discharge light sources traditionally used for fluorescence excitation has been described in several places, including the article entitled “LED fluorescence microscopy in theory and practice” by E. Silk published in the Proceedings and Presentations of the First Annual Citizen Science Conference, Philadelphia, Pa., June 28-29 by the Society for Amateur Scientists, East Greenwich, R.I. (2002), the article entitled “Improvements in fluorescence microscopy allowed by high power light emitting diodes” by G. Mazzini et al. in Current Issues on Multidisciplinary Microscopy Research and Education (ISBN 84-609- 6605-4 ), pp. 181-188, edited by A. Méndez-Vilas and published as FORMATEX Microscopy Book Series by the Formatex Research Centre, Badajoz, Spain (2004), U.S. Pat. No. 6,154,282 issued to Lilge et al. on Nov. 28, 2000 entitled “Semiconductor based excitation illuminator for fluorescence and phosphorescence microscopy”, and PCT patent applications PCT/US01/05107 entitled “Fluorescence microscopy methods and devices using light emission diode” by Barsky et al, published as WO 01/61324 on 16 Feb. 2001 and PCT/IB2004/000976 by M. Angelini entitled “Lighting assembly for a luminescence analysis apparatus, in particular a fluorescence microscope, and luminescence analysis apparatus equipped with such a lighting assembly” published as WO 2004/088387 on 31 Mar. 2004, the contents of all of which are hereby incorporated by reference. All of these references describe means for substituting LEDs for traditional light sources in compound microscopes, and not stereomicroscopes. In most cases (the exception is Silk) the means is to insert the LED source in the same optical path as the original excitation source, thus providing epi-illumination. The adapters must therefore be engineered to fit specific models of microscope. Silk also describes the potential to use LEDs mounted on a table-mounted articulating arm to provide external oblique illumination. Such an implementation is awkward because it occupies valuable bench space and is easily knocked out of position. Moreover, none of the techniques described in these documents are suitable as general purpose adapters.  
         [0029]     A preferred embodiment of the invention will now be described by reference to the accompanying drawings in which, as far as possible, like numbers identify like elements.  
         [0030]      FIG. 1  shows a perspective view of a fluorescence illumination adapter  10 , that is in accordance with a preferred embodiment of the invention, attached to the barrel  12  of a representative, typical stereomicroscope  14 . The stereomicroscope is shown being used to view a specimen  13 . The barrel  12  and the eye pieces  11  contain the stereomicroscope&#39;s imaging optics.  
         [0031]      FIG. 2  shows, in greater detail, a perspective view of a fluorescence exciting illumination adapter  10  that is in accordance with a preferred embodiment of the invention.  
         [0032]     The main body  22  of the fluorescence exciting illumination adapter  10  has a hole  24  that fits around the stereomicroscope barrel  12  (shown in  FIG. 1 ). The main body  22  may have one or more holes  26  that are that are adapted to accommodate thumbscrews (not shown), or other suitable fastening mechanisms, that may, for instance, be threaded, spring-loaded or a sliding friction fit, such that they can be used to removably attach the fluorescence exciting illumination adapter  10  to the stereomicroscope barrel  12  (shown in  FIG. 1 ). There may also be an optional spacer ring  25  to enable the illumination adapter  10  to fit more closely on a stereomicroscope barrel  12  of smaller diameter. Spacer rings  25  (shown in  FIG. 3 ) of varying inner diameters would enable an illumination adapter  10  with fixed inner diameter to work with a wider range of stereomicroscopes  14 .  
         [0033]     In a preferred embodiment, the main body  22  has brackets  28  that hold the illuminator assemblies  30 . Each illuminator assembly  30  may be mounted to a bracket  28  by a machine screw  32  that passes through a hole  34  in the back of the illuminator assembly  30 . Such a mounting arrangement allows the illuminator assemblies  30  to pivot relative to the main body  22 . In this way the while light source and the light source capable of exciting fluorescence are rotatably attached to the rigid frame, or main body  22 , on an axis that is offset and orthogonal to the axis of the cylindrical opening  24 . In this way, the light sources are capable of positional adjustment so as to illuminate an object being imaged by the stereomicroscope for all magnifications that the stereomicroscope is capable of imaging.  
         [0034]     A barrier filter  36  may be removably attached to the underside of the adapter main body  22 . A light shield  38  may also be made of barrier filter material, and may be attached to the back of the central pivoting illuminator element  30  to act as a light shield so that the stereomicroscope user does not directly view the intense illumination on the specimen.  
         [0035]     Each illuminator assembly  30  houses one or more light sources capable of exciting fluorescence. In a preferred embodiment, these light sources capable of exciting fluorescence are suitable Light Emitting Diodes (LED)s capable of exciting fluorescence such as, but not limited to, the Luxeon™ Royal Blue 3W Star LED&#39;s (product code LXHL-LR3C) supplied by Lumileds Lighting, LLC of San Jose, Calif. These LED&#39;s emit up to 340 mW of radiation with peak emission at approximately 455 nm wavelength (in the blue portion of the spectrum) in a Lambertian radiation pattern. In addition each illuminator assembly  30  houses a lens or reflector focusing element to direct the LED illumination onto the specimen.  
         [0036]     The barrier filter  36  is made of material that has spectral characteristics such that it absorbs excitation light that is reflected from the specimen, transmitting only the stimulated fluorescence. By looking at the specimen through the light shield  38  the user may view the fluorescence of the specimen directly, without magnification.  
         [0037]     The excitation radiation from a blue LED may, for instance, be absorbed by a yellow filter material that has maximum absorption in the blue (400 to 500 nanometers) visible light region, and a minimum absorption in the green-to-red (500 to 700 nanometers) visible light region. An example of such filter material suitable for use as a barrier filter  36  and the light shield  38  is the Tiffen™ Yellow 12 “minus blue” glass filter material as supplied by the Tiffen Company of Hauppauge, N.Y. or CYRO 430-7 yellow acrylic material as supplied by CYRO Industries of Rockaway, N.J.  FIG. 3  show the spectral transmission characteristics for a representative yellow filter.  
         [0038]      FIG. 4  illustrates the underside of the main body  22  showing a plurality of small magnets  40  inset into the main body  22 . Suitable magnets include, but are not limited to, rare earth disc magnets such as, for instance, a 6.35 mm diameter by 2.54 mm thick Neodymium-Iron-Boron (NdFeB) disc magnet having a pull force of 971 g (2.14 lb).  
         [0039]      FIG. 5  illustrates the barrier filter element  36  with strips  42  of a metal that can be attracted by the magnets  40 . In normal use, when the barrier filter  36  is brought in close proximity to the underside of the main body  22  the metal strips  42  will be attracted to and held in place by the inset magnets  40 .  
         [0040]      FIG. 6  shows a side cross-sectional view of one of the illuminator assemblies  30 . The body  44  is made of a thermally conductive material such as aluminum to dissipate the heat generated by the LED  46 . A thermally conducting grease (not shown) would be spread between the LED  46  and the illuminator assembly body  44  to assist in heat transfer. A hole  34  is provided at the back of the body  44  for the screw  32  that will be used to attach the illuminator assembly  30  to the main body  22 , and about which the illuminator assembly  30  will pivot. A focusing optic  48  is positioned directly in front of the LED  46  to produce a narrow beam of intense illumination. An optional filter element  50  is positioned in front of the focusing optic  48 . For the LEDs used for fluorescence excitation this filter  50  might be included to limit the range of wavelengths emitted. A compressive ring  52  such as a rubber o-ring is positioned in front of the filter so that when the cover  54  is installed with its mounting screws  56  the optical elements ( 46 ,  48 ,  50 ,  52 ) will be pressed firmly against the illuminator body  44 .  
         [0041]      FIG. 7  illustrates an elevational view of one of the illuminator assemblies  30  mounted in its bracket  28  by the machine screw  32  that serves both as a means attach the elements together and as an axis about which the illuminator assembly  30  is able to pivot. A wave washer  58  or equivalent is positioned between the body  44  of the illuminator assembly  30  and the bracket  28 . This wave washer  58  is in compression so that it presses against the surfaces of the body  44  and the bracket  28  with sufficient force so that when the illuminator assembly  30  is manually pivoted to some angle it will stay there by means of the compressive force.  
         [0042]      FIG. 8  is a schematic drawing showing the adapter connected to a control box  62  and power supply  68 .  
         [0043]     Electrical wires  60  lead from the illuminator assemblies  30  to the control box  62  that includes switch  64  that can select fluorescence excitation, white-light illumination, or a combination of the two and a potentiometer  66  to selectively control the intensity of the white-light illumination. The control box can be powered either by a power supply  68  that plugs into conventional power distribution circuits by means of plug  70 , or by batteries (not shown). In an further embodiment of the invention, the intensity of the white light may be controlled by a means other than a potentiometer, such as, but not limited to well known light dimmer circuits using thyristor and/or triac components.  
         [0044]     Although the invention has been described in language specific to structural features and/or methodological acts, it is to be understood that the invention defined in the appended claims is not necessarily limited to the specific features or acts described. Rather, the specific features and acts are disclosed as exemplary forms of implementing the claimed invention