Patent Description:
This invention was made with government support under grant no. EB021238 awarded by the National Institutes of Health, and grant no. P60016170000198 awarded by the Worcester Science Foundation. The government has certain rights in the invention.

The invention relates generally to a system and methods of viewing a microscope sample using fluorescence microscopy. More specifically, the invention relates to a fluorescence observation system in which a microscope sample may be directly illuminating with an excitation light source without the use of a dichroic mirror.

Fluorescence microscopy plays a major role as a diagnostic tool in many natural-science disciplines. A fundamental principle of fluorescence microscopy is based upon delivery of short-wave excitation light ("excitation light") to a biological sample whereupon the sample itself, or a photoreactive dye mixed with or staining the sample, emits a longer-wave fluorescent light (primary or secondary fluorescence), termed "emission light", upon excitation with the short-wave excitation light. For fluorescence microscopy, secondary fluorescence generally is used to visualize certain specimen structures of stained preparations. It is possible, using fluorescence microscopy, to visualize and/or identify, for example, various pathogens, locate the position of genes, or determine genetic changes in , for example, the DNA that is being examined, or to visualize various proteins and other structures that are formed in a cell.

Various methods of examining a sample using specific fluorescing dyes (so-called fluorophores or fluorochromes) are available to the use. Typical excitation frequencies are in the ultraviolet and visible spectral region. For example, excitation using UV light for the "DAPI" dye, blue light for the "FITC" dye, or green light for the "Texas Red" or "rhodamine" dyes are common.

Short-arc lamps filled with mercury or xenon, or halogen lamps, are used normally as light sources in the illumination systems for fluorescence microscopes. The light sources are most frequently located in a separate lamp housing that is adapted to the microscope. The aforementioned light sources possess a substantially continuous spectrum (UV to IR) that is interspersed with characteristic lines of high intensity. Other excitation light sources include lasers and light emitting diodes. The spectral region appropriate for excitation of a fluorochrome may be selected from the spectral region of the light source by means of various (exchangeable) dielectric filters, called excitation filters. The bandwidth of such excitation filters typically is about from <NUM> to <NUM>.

Microscopes typically include various fluorescence filter systems (so-called filter blocks or filter cubes) so that different stains in a preparation may be visualized. These fluorescence filter systems comprise a mutually coordinated combination of an excitation filter, a dichroic splitter, and a blocking filter. The dichroic mirror reflects the excitation light to the sample, but is transparent to the fluorescent light emitted from the preparation. The blocking filter shields the preparation from scattered excitation light that enters the objective. The various fluorescence filters usually are located on a changing device that may be, for example, a slider or a carousel that is operated either manually and/or in motorized fashion.

In spite of their usefulness, fluorescence microscopes have several disadvantages. For example, such microscopes that may image multiple fluorophores in a sample reactive to different excitation wavelengths may require increasingly more complicated and costly dichroic mirrors in order to image the sample. Alternatively, the imaging of the multiple different fluorophores may require a time-consuming and potentially complex exchange of multiple dichroic mirrors between measurements of each of the fluorophores.

Further, and perhaps more significant than the added expense and complexity of the use of multiple dichroic mirror systems, is the potential for the dichroic mirror to cause an optical aberration in the objective and captured sample image. This may result in a sample image of poor quality, thus requiring special software capable of correcting the image or, alternatively, may require that the experiment be repeated which, in some cases, may not be practical.

Document <CIT> discloses a device for observation by fluorescence of at least one sample placed on a support. This device comprises means of illuminating the sample, an objective for observation of the sample along an observation axis, means of relative displacement of the support with respect to the observation objective, to place the sample on the observation axis, and means of acquisition of the image formed by the observation objective. The illumination means comprises at least one light source.

The observation objective is designed to form an image of the sample when the sample is illuminated, and means of relative displacement of the support with respect to the observation objective.

The device is characterized in that the observation objective is a catadioptric objective and in that the illumination means also comprise means of reflecting light output from the source to the sample, these reflection means being placed between the observation objective and the support. <NPL>, discloses a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. This protocol has streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment.

Document <CIT> discloses an adaptor for a microscope, the adaptor comprising a support part to be received in an objective prism slot of a microscope, a light source element and a mirror located on the support part to receive light from the light source element.

Accordingly, there is a need for a cost-effective system and methods of exposing an objective and sample to light of a certain excitation wavelength for fluorescence observation that circumvents the need for one or more dichroic mirrors in order to capture a high quality of image of the sample of interest. The present invention satisfies this need.

The invention provides for a system and methods of delivering excitation light directly to a microscopy sample without the use of a dichroic mirror that may otherwise cause reflective and/or chromatic aberrations in the objective, and, ultimately, in the captured image of the sample. The dichroic free microscope system uses an apparatus or module that may be attachable to an existing microscope or the module may be made integral to a microscope.

It is provided a microscope system for illuminating a fluorophore as recited in claim <NUM>. The scope for which the protection is sought is provided by the claims.

Embodiments of the invention deliver excitation light directly to the sample through a light delivery path parallel to the optical axis of the objective - through the use of a series of mirrors and, optionally, a diffuser unit - to illuminate a microscope sample.

One certain preferred embodiment of the invention includes an excitation light source, a mirror-based objective configured for use with an objective collar, and one or more excitation tubes optically connected to the excitation light source. The excitation tube may receive and direct an excitation light beam to a focusing element disposed at one end of the excitation tube. The focusing element may be configured to direct the excitation light beam onto a series of mirrors. The series or mirrors includes a first mirror positioned to reflect the excitation light beam received from the focusing element to a second mirror positioned such that is horizontally in line with the first mirror. The second mirror delivers the excitation light beam to a sample positioned above the second mirror.

In other certain embodiments of the invention, a light diffuser unit may be positioned between the second mirror and the sample. The light diffuser unit may be configurable to adjust the illumination profile of the excitation light beam to deliver a homogenous distribution of power across the field of view of the sample.

Embodiments of the invention include also a mirror positioning adapter coupled to the objective and objective collar. The mirror positioning adapter is adjustable along several axes, and may include, for example, a sliding mechanism adjustable along each of the x, y, z axis to permit the user to adjust the positioning and angle of the reflective element or focusing elements to which it is mechanically coupled in order to direct the excitation light to the sample via a second mirror and/or diffuser unit. The mirror positioning adapter is adjustable by the user either through manual or automated means.

Advantageously, embodiments of the invention make use of a particular geometry and a long working distance of the mirror-based objective - that is, the clearance between the objective enclosure and the sample or focus plane - to position one or more small mirrors that may be used to deliver excitation light to the sample independent of a dichroic mirror. The use of such mirrors may allow also the use of any and all excitation wavelengths within the reflective range (i.e., wavelength) of the small mirrors that is not possible using a dichroic mirror.

Advantageously, the removal of the dichroic mirror - such as during the use of a mirror-based objective to either directly form an image of the sample or used with a curved mirror such as a 'tube-lens' to form the image - eliminates any non-orthogonal dispersive materials in the emission path. This makes the system fully achromatic.

Advantageously, the optical elements (e.g., mirrors) do not reduce the amount of light collected through the sample-facing aperture of the objective.

Advantageously, use of the present invention may allow for custom shaping of the excitation light profile as it passes into the sample through the use of a diffuser positioning above the second mirror (on top of the objective). The profile of the excitation light may be shaped into, for example, a flat 'top-hat' profile, rather than the traditional Gaussian profile, to allow a more homogenous excitation of fluorophores across the field of view.

Advantageously, embodiments of the present invention use a mirror system to deliver light of any wavelength to a sample without the need to change or remove an optical element (e.g., dichroic mirror) when moving between measurements of multiple fluorophores in a sample.

Advantageously, embodiments of the present invention permit the objective and the excitation unit (e.g., focusing lenses, reflective mirrors, diffuser unit) to move in unison during focus adjustment.

The present invention and its attributes and advantages will be further understood and appreciated with reference to the detailed description below of presently contemplated embodiments, taken in conjunction with the accompanying drawings.

The preferred embodiments of the invention will be described in conjunction with the appended drawings provided to illustrate and not to the limit the invention, where like designations denote like elements, and in which:.

The present invention generally is directed to a system and methods that use an apparatus to deliver excitation light directly to a microscope sample - by bypassing the objective and without the use of a dichroic mirror that may otherwise cause chromatic aberrations - that may be used with mirror-based objective lenses.

<FIG> illustrates a typical microscope <NUM> that uses a dichroic mirror <NUM> (hereafter referred to as a "DM microscope") to deliver excitation light <NUM> from an excitation light source to a microscope sample positioned above objective <NUM>. In some of the figures, certain parts are "cut-away" to show the pathway of the light.

Suitable excitation light source for use with the invention may include any light source that may emit light in the desired wavelength such as a laser, light emitting diodes, a bulb (e.g., mercury-arc, xenon-arc, or metal-halide), or other white light or full light spectrum sources. In certain preferred embodiments, the excitation light source is a bulb emitting multiple wavelengths capable of exciting fluorophores.

The excitation light source (e.g., a laser) may be optically coupled to the DM microscope through the use of one or more reflective elements positioned within the reflective element housing <NUM>. One or more support members <NUM> may support the reflective element housing <NUM>. The reflective element housing <NUM> may include one or more adjustment elements <NUM> configurable to adjust the angle and direction of each reflective element positioned within the reflective element housing <NUM>. The excitation light beam <NUM> may be directed from the reflective elements in the reflective element housing <NUM> to the filter cube housing <NUM> through the excitation tube lens <NUM>. The excitation tube lens <NUM> may comprise one or more convex or concave lenses that may be used to focus and/or adjust the path of the excitation beam <NUM>. The excitation light beam then may pass through one or more excitation filters <NUM> positioned in the filer cube housing that may be used to select the wavelength of the excitation light beam that illuminates the sample. The excitation filters <NUM> may be manually removable and replaceable through, for example, a slot in the filter cube housing, depending on the excitation wavelength and fluorophores in the sample. Alternatively, the excitation filters may be movable within the filter cube housing through automated means such as a turret.

After passing through the excitation filters, the excitation light beam encounters a dichroic mirror <NUM> positioned in the path of the excitation light beam. The dichroic mirror <NUM> may be adjustable, either manually or through automated means (e.g., interface with a controller), to any angle in relation to the excitation light beam path. For example, the dichroic mirror <NUM> may be positioned from about a <NUM>-degree angle to about a <NUM>-degree angle relative to the excitation light beam path. The dichroic mirror <NUM> may reflect a portion of the excitation light beam <NUM> through a series of manual focusing components <NUM> and/or pi-foc piero focusing elements <NUM> to a reflective objective <NUM> and objective collar <NUM>. The excitation light beam may then pass through the objective <NUM> and illuminate the sample, causing a fluorophore in the sample to emit an emission light beam <NUM> (i.e., light of a different wavelength than the excitation light. The objective <NUM> may capture the emission light and direct it back through the dichroic mirror <NUM> and, optionally, one or more emission filters <NUM> positioned below the filter cube housing <NUM>. The emission light then may be directed to one or more mirrors (not shown) such as, for example, a parabolic mirror, positioned within the mirror housing <NUM> and in the path of the emission beam light. The mirrors reflect the emission beam through the emissions tube <NUM> where the emission light is collected by an imaging capturing device such as a camera coupled to the microscope to display an image on a suitable viewing apparatus such as a computer screen or the like.

In contrast, a dichroic-free microscope (hereafter "DF microscope") is not dependent upon the dichroic mirror to deliver an excitation light beam to the sample and objective. Instead, a DF microscope may use an excitation light source coupled to a series of mirrors to directly illuminate a sample while bypassing the objective and the dichroic mirror entirely. Thus, the excitation light that is delivered to the sample is essentially achromatic (since the light does not pass through the dichroic mirror and/or filters). Further, the use of a DF microscope eliminates many of the reflective and chromatic aberrations introduced into the system by the tilted and/or curved glass surfaces or surfaces having a certain flatness and/or variation in thickness in the excitation/emission light path. For example, imperfections of the dichroic mirror may impact the wave front of the emission light that may result in optical aberrations.

<FIG> and <FIG> illustrate one certain preferred embodiment of a DF microscope <NUM>, <NUM>. The DF microscope <NUM>, <NUM> may include many of the components of a DM microscope such as described in <FIG>: a reflective objective <NUM>, <NUM> and objective collar <NUM>, <NUM>, pi-foc focusing element <NUM>, <NUM>, manual focusing element <NUM>, <NUM>, filter cube housing <NUM>, <NUM>, dichroic mirror <NUM>, <NUM>, emissions filters <NUM>, <NUM>, parabolic mirror support <NUM>, <NUM>, and parabolic mirror <NUM>, <NUM>. In addition to these components, the DF microscope may include further an excitation light source (not pictured) coupled to an excitation beam positioner <NUM>, <NUM>, that comprises a housing and one or more reflective elements <NUM>, <NUM> that is supported by mounting plate <NUM>. The excitation beam positioner <NUM>, <NUM> directs the excitation light beam through the excitation beam tube <NUM>, <NUM>, to the focusing element housing <NUM>, <NUM>. The focusing element housing <NUM>, <NUM> may include one or more focusing elements <NUM> (e.g., lenses, mirrors, curved mirrors) that may be used to focus or otherwise adjust the excitation light beam to impact a reflective element <NUM> and/or the sample.

Embodiments of the invention include also a mirror position adapter <NUM>, <NUM> mechanically connected to the reflective element <NUM> that may be used to adjust the reflective element <NUM> so that the excitation light beam impacts a second mirror, optionally including a diffuser unit <NUM> and is then channeled to the sample of interest. Support members <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> may provide structural support for the focusing element housing <NUM>, <NUM>, excitation beam tube <NUM>, <NUM>, mirror position adapter <NUM>, <NUM>, objective <NUM>, <NUM> and objective collar <NUM>, <NUM>, and diffuser unit <NUM>.

<FIG>, <FIG>, and <FIG> illustrate the objective, illumination components, and supporting structures of one preferred embodiment of the invention. The excitation light beam (shown travelling through the excitation tube) may be directed from the excitation light source, through one or more mirrors positioned within the excitation beam positioner <NUM>, and into the excitation beam tube <NUM> that is coupled to the objective collar. Once in the excitation beam tube <NUM>, the excitation light encounters focusing elements <NUM> positioned in the focusing element housing <NUM>, <NUM>. The focusing element housing may be positioned below mounting plate <NUM>, <NUM> that also may function as a support for the excitation beam tube <NUM>, <NUM>. The mounting plate <NUM>, <NUM> and support <NUM> also may connect and support the mirror position adapter <NUM>, <NUM> and objective collar <NUM>, <NUM>. The excitation light tube <NUM> also may be configured to receive an alignment tool to align the excitation beam with the optical axis of the microscope and/or the optical axis of the mirror.

The excitation light may then pass through the focusing element <NUM> (e.g. a lens or mirror) positioned within the focusing element housing <NUM>, <NUM>. The focusing element <NUM> may be, for example, a convex lens and may converge the incoming parallel excitation light beam in order to control the size of the illuminated area of the sample. The focusing element <NUM> may adjust focus of the excitation light and/or the excitation "spot" where the beam impacts the sample by moving the focusing element <NUM> or focusing element housing <NUM>, <NUM>, for example, either upward or downward in a linear fashion in the mounting plate <NUM>, <NUM>.

The focusing element <NUM> delivers the excitation light beam parallel to the focal plane of the sample through the use of reflective elements <NUM>, <NUM>, <NUM>. <NUM> (or any small reflective surface or light steering material, e.g. a crystal, mirror, prism) each having a length of about <NUM> millimeters to about <NUM> millimeters dependent upon the objective with which the reflective elements are used. Alternatively, embodiments of the invention may deliver the excitation light beam into the mirror system without the use of the focusing element <NUM>. The reflective elements <NUM>, <NUM>, <NUM>, <NUM> may be positioned and angled to reflect the excitation light beam as needed (e.g., <NUM>°, <NUM>°).

The relatively long working-distance (~<NUM>) of the objective <NUM>, <NUM> used to image emission light from a fluorophore may allow the placement of one or more of the reflective elements (e.g., <NUM>, <NUM>) to direct the excitation light beam from the focusing element <NUM> to the sample. Preferably, a first reflective element <NUM>, <NUM> may be disposed on or within the objective collar <NUM>, <NUM> between the objective <NUM>, <NUM> and the sample (e.g. on the spider described below). A second reflective element <NUM>, <NUM> is positioned in line with the first reflective element <NUM>, <NUM>. The second reflective element <NUM>, <NUM> is supported by a mirror positioning adapter <NUM>, <NUM> connected to the objective collar <NUM>, <NUM>. The mirror positioning adapter <NUM>, <NUM> permits movement of the mirror in the x, y, and z directions. One embodiment of the mirror positioning adapter may include, for example, a sliding mechanism adjustable along each of the x, y, z axis to enable rotation and movement of the second reflective element <NUM>, <NUM> relative to the first reflective element <NUM>, <NUM> and may be used to focus and/or adjust the excitation light beam. The mirror position adjustment may be either manually or robotically controllable by mechanically coupling x, y, and z translation motors through or more control systems. Preferably, each of the reflective mirrors are angled at <NUM>° to direct the incoming light at <NUM>°.

<FIG> illustrates one certain embodiment of the invention that may include also a diffuser unit <NUM> positioned between the first reflective element <NUM> and the sample <NUM>. The diffuser unit <NUM> may include a diffuser element <NUM> supported by a diffuser support component <NUM> disposed on or within the objective collar <NUM>. The diffuser element <NUM> may be used to create a relatively homogenous illumination profile <NUM> (e.g., a 'top-hat' profile) of excitation beam <NUM> that may be determined by the user rather than the traditional Gaussian profile which results in a non-uniform power density across the field of view. Various diffuser elements may be constructed or purchased to provide different illumination profiles. The diffuser <NUM> may be constructed from a translucent, transparent, or semi-transparent plastic material, such as polymethyl methacrylate (PMMA). The surface of the diffuser <NUM> may be constructed to have a rough or smooth surface in order to achieve the desired optical property. The diffuser element <NUM> also may be constructed to have a certain shape that will provide a certain illumination profile <NUM>, <NUM> (e.g. "top-hat" illumination profile).

<FIG> illustrates a top view of one preferred embodiment of the invention showing the excitation light beam <NUM> being directed by a first reflective element <NUM> to a second reflective element <NUM>. The second reflective element <NUM> then directs the excitation beam upward and parallel to optical axis of the objective directly to the sample, or, first through a diffuser unit <NUM> positioned above spider element <NUM> - that itself is part of objective collar <NUM> - and onto the sample. Holes <NUM> may be configured to accept various alignment tools to facilitate alignment or other manipulations of the reflective elements.

<FIG> illustrate one embodiment of a spider <NUM> that comprises one or more support structures (arms, spokes, beams, etc.) arranged to extend outward in a plane from a center point <NUM> to objective collar <NUM>. Typically, the spider <NUM> supports a secondary mirror positioned on an inner surface of the center point <NUM>. The support structures <NUM> may act as a central obscuration (blocked area) that causes a reduction of the contrast for low to mid spatial frequencies. The support structures <NUM> also may cause a faint diffraction pattern that may reduce the quality of the image of the sample. Advantageously, placement of the diffuser unit on the center point <NUM> of the spider will not lead to further obstruction of the light compared to the placement of the diffuser in another position that interferes with the light path.

After the excitation beam has been directed to the sample, one or more fluorophores in the sample may emit an emission beam that is captured by the objective and directed through the dichroic mirror. The emission light is then captured by one or more imaging capture device coupled to the microscope through the emissions tube <NUM>. Preferably, the image capturing device is a high resolution, color, digital camera operating at a high-resolution and a high data rate. Images captured by the camera are directed via control electronics, such as a camera link card, to one or more control processors. As is well known by those of ordinary skill in the art, a camera link card may interface with digital cameras supporting the particular protocol and physical interface. Other protocols and physical interfaces are also contemplated in the context of the invention, and one particular interface described is not to be taken as limiting in any way.

A control processor, which may be implemented as a small platform computer system, provides the data processing and platform capabilities for hosting an application software program suitable for developing the necessary command and control signals for operating the microscope system. Control processor includes specialized software or circuitry capable of performing image processing functions. For example, control processor may perform image analysis and obtain measurements of contrast, entropy, sharpness, etc. Control processor may also contain specialized software or circuitry capable of manipulating and combining digital images. The control processor may be able to receive and interpret commands issued by a system user on a conventional input device, such as a mouse or a keyboard, and is further able to convert user defined commands into signals appropriate for manipulating the various components of the microscope system.

The control processor is typically coupled to the microscopy system through an interface, such as a serial interface, a Peripheral Component Interconnect (PCI) interface, or anyone of a number of alternative coupling interfaces, which, in turn, defines a system interface to which the various control electronics operating the microscope system are connected.

Claim 1:
A microscope system for illuminating a fluorophore comprising:
an excitation light source;
a microscope comprising:
a mirror-based objective (<NUM>, <NUM>) supporting an objective collar (<NUM>, <NUM>);
a tube configured to receive an excitation light beam directed parallel to an optical axis of said mirror-based objective (<NUM>, <NUM>), said tube connected to said objective collar (<NUM>, <NUM>),
a first reflective element (<NUM>, <NUM>, <NUM>, <NUM>) positioned to reflect the excitation light beam (<NUM>), said first reflective element (<NUM>, <NUM>, <NUM>, <NUM>) disposed in a reflective element positioning adapter (<NUM>, <NUM>) that is movable in several axes, said reflective element positioning adapter (<NUM>, <NUM>) connected to said objective collar (<NUM>, <NUM>); and
a second reflective element (<NUM>, <NUM>, <NUM>) positioned horizontally and in line with said first reflective element (<NUM>, <NUM>, <NUM>) and disposed on a center support element, said second reflective element (<NUM>, <NUM>, <NUM>) configured to reflect the excitation light beam into a sample to cause fluorescence of the fluorophore in the sample.