Optical system

An optical system is provided for achieving enhanced rejection of scattered excitation light and superior signal-to-noise performance when reading microplate wells. The optical system uses an axial configuration in which the excitation beam incident upon the sample propagates along the axis of the microplate well. Excitation light from a light source, such as a lamp or fiber optic bundle, is collimated into a beam using a lens. A reflective pick-off mirror is then used to reflect the collimated excitation beam upward along the well axis. A focusing lens, with a diameter exceeding the diameter of the collimated excitation beam, is used to focus the excitation beam in the well. The same broad lens is used to collimate the emitted fluorescent light, of which a large percentage propagates axially past the pick-off mirror towards a second focusing lens that focuses the emission beam onto the face of a fiber optic bundle. The emitted light is later filtered and detected using at a position that is optically shielded from the aforementioned optical system. The optical system is incorporated into a microplate reader or automated assay instrument in order to provide a compact assembly for sensitive fluorescence measurements either above or below the microplate. The optical system further enables the simultaneous measurement of absorbance and fluorescence in a compact optical configuration.

FIELD OF THE INVENTION

The present invention is related to an optical system for achieving enhanced rejection of scattered excitation light and superior signal-to-noise performance when reading microplate wells and more particularly the present invention relates to an optical system which enables the simultaneous measurement of absorbance and fluorescence in a compact optical configuration.

BACKGROUND OF THE INVENTION

Optical measurements are routinely used to detect and quantify the presence of species in chemical and biological samples. The most common forms of optical measurement are absorption, fluorescence, and luminescence. In absorbance measurements, the degree of attenuation caused by optical absorption is related to the concentration of the species of interest. Fluorescence measurements involve exciting the sample via short-wavelength optical radiation and measuring the optical power of longer-wavelength fluorescence. Luminescence measurements rely on the measurement of light emitted by the sample without the presence of excitation light, most often caused by a chemical reaction.

In modern analytical instruments, small sample volumes are arranged in a multiple-well plate known as a microplate, usually comprised of 96, 384 or 1536 individual wells. A typical instrument optically scans the wells and detects optical power. The optical power is then related to the sample concentration using a known functional relationship. Fluorescence is most commonly used as a sensitive means to detect very low sample concentrations and maintain a wide dynamic range.

Despite the large number of patents pertaining to fluorometric instruments, little attention has been paid to the elimination of stray excitation light, which can cause significant signal degradation. Stray light can arise from a multitude of sources, including lens surfaces, microplate window surfaces, and scattering within the optical system. However, the dominant source of stray light in a microplate fluorometer is usually the liquid meniscus at the sample-air interface. This source of stray light is particularly difficult to reject since the meniscus can vary in position (due to different sample volumes) and curvature (due to different surface properties). Most importantly, a spatially extended beam impinging on the meniscus results in a wide angular bandwidth upon reflection, making it difficult to achieve rejection by simple baffling alone. One patent that has attempted to improve upon the prior art in this regard is listed below.

U.S. Pat. No. 6,316,774, titled “Optical System for a Scanning Fluorometer”, discloses an optical instrument for the measurement of absorbance and fluorescence from microplates. The patent teaches a method of measuring fluorescence from above and below a microplate well using elliptical reflective mirrors that are arranged in a manner in which the collection of excitation beam scatter is reduced. Unfortunately, the invention only succeeds in eliminating the collection of scattered excitation light for a very limited range of sample volumes and meniscus curvatures.

It would be very advantageous to provide an optical system that could broadly be used to reduce interference arising from liquid scattering of excitation light from a liquid meniscus in any type of optical system used for optical analysis of liquids contained in vessels, which advantageously could be integrated into a microplate reader or automated assay instrument in order to provide a compact assembly for sensitive fluorescence measurements that avoids or reduces the interference of scattered light from the liquid meniscus.

SUMMARY OF THE INVENTION

Accordingly, an invention is provided in which a compact optical system is used to measure fluorescence from a microplate well with exceptional rejection of scattered excitation light. This is accomplished using an axial configuration in which the excitation beam incident upon the sample propagates along the axis of the microplate well. Excitation light from a source, such as a lamp or fiber optic bundle, is collimated into a beam using a lens. A reflective pick-off mirror is then used to reflect the collimated excitation beam upward along the well axis. A focusing lens, with a diameter exceeding the diameter of the collimated excitation beam, is used to focus the excitation beam in the well. The same broad lens is used to collimate the emitted fluorescent light, of which a large percentage propagates axially past the pick-off mirror towards a second focusing lens that focuses the emission beam onto the face of a fiber optic bundle. The emitted light is later filtered and detected using methods known to those skilled in the art at a position that is optically shielded from the aforementioned optical system. The optical system is incorporated into a microplate reader or automated assay instrument in order to provide a compact assembly for sensitive fluorescence measurements either above or below the microplate.

Thus, the present invention provides an optical system for measuring fluorescence from a liquid contained in a vessel, comprising:

a) a light source and a first lens for collimating excitation light from said light source into a beam of excitation light, a fully reflective mirror positioned to receive and redirect the beam of excitation light in a first direction along an axis of a vessel located in a vessel holder located on one side of said fully reflective mirror, a second lens positioned along said axis on said one side of said vessel between said fully reflective mirror and said vessel for focusing said beam of excitation light onto said vessel and collimating light emitted from a liquid in said vessel;

b) a third lens symmetrically disposed with respect to said axis of said vessel and on a second side of said fully reflective mirror, for focusing light collimated by said second lens onto a first detection means; and

c) said second lens having a diameter larger than a cross sectional area of said fully reflective mirror transverse to said axis such that excitation light scattered from refractive surfaces within the vessel including a liquid meniscus is rejected by said fully reflective mirror, but some fluorescent light emitted by the liquid in said vessel bypasses the fully reflective mirror and is focused onto the detection means by said third lens.

The optical system may include a second detection means positioned relative to said vessel so that the optical power of the excitation beam that propagates axially through the microplate well can be measured on the opposing side of the microplate in order to provide a measurement of absorbance. A dual mode system is therefore enabled by the invention in which absorbance and/or fluorescence can be measured simultaneously.

The size of the optical source and the magnification of the excitation beam path optics may be chosen in such a way as to produce a small focused spot in the center of the microplate well. This small spot, preferably less than 2 mm, causes the excitation beam to encounter only the zone within the center of the liquid meniscus having a near-axial surface normal. The axial surface normal of the central meniscus causes the angular bandwidth of the reflected excitation cone to be narrow, enabling rejection via apertures within the optical system.

A limiting aperture may be used to produce a low numerical aperture excitation beam. A low numerical aperture is desirable for two distinct reasons. Firstly, the low numerical aperture makes the excitation beam more amenable for absorption measurements, since the optical path through the well is made more columnar and less conical. Secondly, the lower angular bandwidth of the excitation beam incident on the well also serves to lower the angular bandwidth of the scattered excitation light cone produced by the meniscus. The limiting aperture also helps to eliminate multiple reflections caused by the lens collimating the excitation beam from the light source or fiber optic bundle.

The pick-off mirror is preferably chosen to have a diameter larger than that of the collimated excitation beam in order to reject a substantial amount of the low numerical aperture component of the scattered excitation light. The rejection of this component of the scattered excitation light via a mirrored surface removes a large fraction of the scattered excitation light from the optical system.

A shadow disc, comprising a thin opaque disc that preferentially absorbs light and reflects diffusely, may be placed immediately above the second focusing lens (the lens that focuses the emitted light) in order to stop scattered excitation light from reaching the focus. As a result of the aforementioned aspects that decrease the angular bandwidth of the scattered excitation light, the scattered excitation cone (the outer regions of which are produced by the meniscus) collimates to a diameter that can be less than that of the first lens (the lens that focuses the excitation beam and collimates the emitted light). By choosing a shadow disc that has a diameter that is slightly larger than the diameter of the collimated scattered excitation beam but less than that of the upper lens, the scattered excitation light is very effectively prohibited from directly scattering to the focus. Finally, since the scattered excitation beam is in many cases slowly converging after propagating through the first lens, a free propagation path between the first lens and the shadow disc can further increase the power of the collected emission light relative to the scattered excitation light.

Thus, in another aspect of the invention there is provided an optical system for measuring fluorescence, comprising:

a) a light source and a first lens for collimating light from said light source into a beam of light, a reflective mirror positioned to receive and redirect the beam of light in a first direction along an axis of a vessel located on one side of said reflective mirror, a second lens positioned along said axis on said one side of said vessel between said reflective mirror and said vessel for focusing said beam of light onto said vessel and collimating light emitted from a liquid in said vessel;

b) a third lens symmetrically disposed with respect to said axis of said vessel and located on a second side of said reflective mirror, for focusing light collimated by said second lens onto a first detection means; and

c) a shadow disc symmetrically disposed with respect to said axis located between the said reflecting mirror and said third lens, wherein a diameter of said shadow disc is greater than that of a cross sectional area of said fully reflective mirror transverse to said axis but less than the diameter of said second lens such that excitation light scattered from refractive surfaces within the vessel including a liquid meniscus is rejected by said shadow disc, but some fluorescent light emitted by the liquid in said vessel bypasses the fully reflective mirror and is focused onto the detection means by said third lens.

The present invention also provides an embodiment of an optical system in which the aforementioned lenses are replaced with mirrors that simplify the design and eliminate chromatic aberrations. The low numerical aperture lens that had collimated the source and the reflecting mirror may be replaced with a single concave mirror that refocuses the excitation beam into the liquid containing vessel along an axis of the vessel. The upper focusing lens is replaced with an off-axis parabolic mirror. This mirror collimates the emitted light and reflects it in a direction normal to the well bottom. This mirror has a hole cut through it in order to pass the focusing excitation beam that is directed along the well axis. An aspheric lens may then be used to focus the light emitted from the liquid onto a detector, or a fiber bundle optically coupled to the detector. In front of the aspheric lens there may be included a shadow disc, which again serves to reject the narrow beam of collimated (or slowly converging) scattered excitation light. The aspheric lens may also be replaced with an on- or off-axis parabolic mirror.

Thus, there is provided an optical system for measuring fluorescence from a liquid contained in a vessel, comprising:

a) a light source and a first concave mirror for directing and focusing excitation light beam from said light source onto said vessel, along an axis of said vessel located in a vessel holder;

b) a second concave mirror for collimating and redirecting light emitted from the liquid in said vessel along another axis, said second concave mirror being located between said vessel and said first concave mirror, said second concave mirror including a hole through which the excitation light beam reflected by the first concave mirror passes;

c) a directing and focusing means for directing and focusing light collimated and redirected from said second concave mirror onto a detection means; and

d) wherein excitation light scattered from refractive surfaces within the vessel including a liquid meniscus passes through said hole, but some fluorescent light emitted by the liquid in said vessel is reflected by the second concave mirror and is focused onto the detection means by said directing and focusing means.

The present invention also provides a method for measuring fluorescence from a liquid contained in a vessel, comprising the steps of:

a) collimating light from a light source into a beam of excitation light and directing the collimated beam of excitation light toward a fully reflective mirror which receives the collimated beam of excitation light and redirects it in a first direction along an axis of a vessel located in a vessel holder located on one side of said fully reflective mirror, focusing said collimated beam of excitation light onto said vessel and collimating light emitted from a liquid in said vessel and directing the collimated light emitted from the vessel in a direction 180 degrees to said first direction back along said axis, wherein said step of focusing said collimated beam of excitation light onto said vessel and collimating light emitted from a liquid in said vessel is performed using a first focusing and collimating means; and

b) focusing the collimated light emitted from the vessel onto a first detection means located on another side of said fully reflecting mirror using a second focusing means, wherein said first focusing and collimating means has a diameter larger than a cross sectional area of said fully reflective mirror transverse to said axis such that excitation light scattered from refractive surfaces within the vessel including a liquid meniscus is rejected by said fully reflective mirror, but some fluorescent light emitted by the liquid in said vessel bypasses the fully reflective mirror and is focused onto the detection means by said second focusing means.

The present invention also provides a method for measuring fluorescence from a liquid contained in a vessel, comprising the steps of:

a) directing and focusing excitation light from a light source onto a vessel along an axis of said vessel using a first concave mirror;

b) collecting, collimating and redirecting light emitted from the liquid in said vessel along another axis using a second concave mirror, said second concave mirror being located between said vessel and said first concave mirror, said second concave mirror including a hole through which the excitation light beam reflected by the first concave mirror passes; and

c) directing and focusing light collimated and redirected from said second concave mirror onto a detection means, wherein excitation light scattered from refractive surfaces within the vessel including a liquid meniscus passes through said hole, but some fluorescent light emitted by the liquid in said vessel is reflected by the second concave mirror and is focused onto the detection means by said directing and focusing means.

A further understanding of the functional and advantageous aspects of the invention can be realized by reference to the following detailed description and drawings.

DETAILED DESCRIPTION OF THE INVENTION

Referring toFIG. 1, there is a schematic of a conventional optical system10for measuring fluorescence from a microplate well or any other type of liquid sample containing vessel. InFIG. 1A, an optical source1, usually either a filtered light source or a fiber optic bundle delivering filtered light, is collimated by lens2and reflected to the microplate well5by beamsplitter3. A second lens4focuses the excitation light onto the microplate well, where it is focused into the fluorescent sample liquid6with meniscus7. InFIG. 1B, the emitted light from the sample15is collimated by lens4, propagates through beamsplitter3and is focused with lens8to a focal point9. If no emission filter is used between beamsplitter3and lens8, then focus9is usually coincident with the aperture of a fiber bundle that delivers the emitted light to a remote filtering and detection location. Otherwise, a detector is placed at the focus9and the optical power of the emitted light is measured.

It will be appreciated that while the present invention is being described using liquid vessels which are microplate wells, the optical system disclosed herein is may be used with any type of liquid containing vessel in which scattering of light by the liquid meniscus may be problematic.

FIGS. 2A and 2Bshow the geometric paths taken by excitation beam rays as they scatter off of the reflective surfaces of the optical system shown inFIG. 1.FIG. 2Aillustrates excitation beam scattering from the side view andFIG. 2Bprovides an overhead perspective.FIGS. 2A and 2Bclearly reveal the meniscus7as the dominant source of scattering in this configuration. Although the well bottom and focusing lens4produce a retro-reflection of the incident beam along its incoming path, the curvature of the meniscus7causes the scattered rays to fan out over a wide angular zone. The meniscus therefore acts as a virtual source of excitation light within the fluorescent zone of the liquid sample6. Even if one blocks the primary beam path through the beamsplitter and collects light emitted in the outer angular zones, a large percentage of scattered excitation light will still corrupt the measured signal.

An alternative prior art optical system that improves upon that ofFIG. 1is shown inFIGS. 3A and 3B. This optical system employs a non-axial configuration in order to reduce the amount of scattered excitation light that reaches the detector. Both the excitation axis and emission axis are oriented at an angle of 30° relative to the well axis, and the excitation and emission axes are orthogonal when projected onto the horizontal plane. The source light30is collimated and focused onto the well7by lenses31and32, respectively. Lenses33and34collect and re-image the fluorescent light emitted by the well. In this configuration, the portion of the excitation beam that scatters off of the well bottom is advantageously directed away from the solid angle subtended by the detector.

Although the design is well suited for eliminating the collection of scattered excitation light from flat surfaces, it fails to sufficiently discriminate between emitted and scattered light when a curved surface is present.FIGS. 3A and 3Billustrate the poor performance obtained with a curved meniscus from the perspectives of aFIG. 3Ashowing the side view andFIG. 3Bshowing the overhead view. Although most rays scatter away from collimating lens33, a sizable fraction is scattered within the collection solid angle. These rays are eventually imaged onto the focus9and result in a decreased signal-to-noise ratio.

Referring now toFIGS. 4A,4B and4C, there is shown an optical system in accordance with the present invention to obtain a dramatic reduction in the amount of collected scattered excitation light.FIG. 4Aillustrates the propagation of the excitation beam through the system without considering scattering. Lens41collimates the light from light source40into a beam that is passed through aperture44. This aperture serves to reduce the diameter of the collimated beam and thus lower the numerical aperture of the beam as it is focused onto the microplate well. The fully reflective pick-off mirror42redirects the beam upwards along the axis of the microplate well. The excitation beam is then focused using lens43onto the microplate well5. The beam passes through the well and is detected above the well by absorbance detector48. InFIG. 4B, the collection of emitted rays from the optically excited fluorescent liquid6is shown. Although many of the small-angle emitted rays49are rejected by the pick-off mirror42, the majority of the emitted power that is collected by the upper lens43passes the pick-off mirror. The shadow disc45further attenuates the collimated emission beam, but allows a thin collimated ring to pass onto the lower lens46, which focuses the emission beam to focal point47. The diameter of the lower lens46is preferably greater than or equal to the diameter of the upper lens43.

In a preferred embodiment, the light source40is preferably a narrow band source, such a fiber optic bundle that delivers light from a remote source and filtering subsystem. Alternatively, a broadband light source can be filtered within the inventive optical system by placing a filter between the aperture44and the fully reflective pick-off mirror42. Also, in a preferred embodiment, a light conduit (not shown) resides at the focal point47. This conduit can be, for example, a fiber optic bundle for the delivery of the collected fluorescent light to a remote filtering and detection subsystem. Alternatively, a filter can be placed between the upper lens43and the lower lens46, whereby the filtered fluorescence light is detected locally by a detector placed at the focus47.

The advantageous aspect of the invention is shown inFIG. 4C, in which the scattered rays from the excitation beam are depicted. The majority of the scattered rays are produced by the liquid meniscus7, which again acts as a source of a cone of scattered light. All scattered rays are collimated by the lens43. However, since the mirror42is now entirely reflecting rather than partially reflecting, a large portion of the scattered rays are deflected horizontally and do not reach the focal point47. The scattered rays produced by the upper surface of lens43and the bottom of the microplate well7are thus entirely removed by the pick-off mirror42. The large-angle scattered rays (produced by the meniscus) that do succeed in bypassing the pick-off mirror42are blocked by shadow disc45, which preferably absorbs all rays that impinge upon it. The only excitation rays that do travel beyond the shadow disc45originate at the lower surface of the upper lens43. Since these rays are not in a conjugate image plane of the microplate well, they are not imaged onto the final focus47and are therefore of no consequence.

It can also be seen inFIG. 4Cthat the collimated scattered excitation rays are not perfectly collimated, but slightly converge after passing through lens43. It is therefore evident that placing the shadow disc45further away from the lens43improves the performance of the system in that the ratio of the detected emission power to the detected scattered excitation power is enhanced. This ratio forms a useful figure of merit for the optical system and is henceforth referred to as the signal-scattering extinction ratio (SSER). In a preferred embodiment, the spatial separation between the lens43and the shadow disc45is greater than or equal to the diameter of the lens43.

In order to obtain a very high SSER over a wide range of sample volumes (i.e. meniscus heights) and meniscus curvatures, the size and angular bandwidth of the incident excitation beam must be carefully designed. In particular, the size of the beam focus must be kept to a minimum so that the spatial extent of the beam only samples the central region of the meniscus. A broad, extended beam will sample a wide range of curvatures, producing a deleterious wide cone of scattered excitation light. A tight focus, with a size preferably less than 25% of the diameter of the meniscus, will produce a reflection with a relatively narrow angular bandwidth. In this context, the diameter of the meniscus is defined as the perimeter of the meniscus at its point of contact with the inner surface of the vessel. In the case of a 96-well microplate, the size of the beam focus is preferably 2 mm or less. This narrow cone of scattered excitation light is more amenable to complete rejection by the shadow disc45.

The requirement that the spatial extent of the beam be less than 2 mm over a wide range of fluid volumes also implies that the numerical aperture of the incident beam also be low. A low numerical aperture results in a beam that is only weakly converging and diverging on opposite sides of the focus. The numerical aperture of the excitation beam is preferably less than 0.20 in order to obtain a high SSER. Such a low numerical aperture also facilitates the measurement of absorption, which is preferably performed with a collimated beam passing through the sample. In practice, a low-numerical aperture beam is also useful for absorption measurements. The combination of a small focus and low numerical aperture provides a near-collimated beam within the well that is not clipped by the sides of the well upon transmission.

A further requirement for a high SSER is the need for accurate placement of the excitation beam focus within the center of the microplate well5. A deviation of the focal point from the well center will result in the beam encountering a point on the meniscus where the central surface normal is not directed along the well axis. This in turn causes the scattered excitation cone to be deflected along a non-axial direction and some scattered rays may bypass the shadow disc45and reach the final focus47. A positional tolerance of ±0.25 mm is preferred for optimal performance. This requirement also assists in providing an optimal absorbance measurement, since deviations of the beam relative to the meniscus center can cause path length variations that produce absorption errors.

The diameter of the shadow disc45must be sufficiently large to intercept all excitation rays that are directly scattered by the meniscus. The shadow disc is preferably made of a material that absorbs all the light that impinges upon it.

A maximum limit for the diameter is set by the diameter of the upper lens43, which defines the width of the collimated emission light. The diameter of the shadow disc is preferably at least 75% of the diameter of the lens43. Although this attenuates the emission power, the attenuation is proportion to the square of the diameter ratio, resulting in a loss of only approximately 50% or 3 dB. The corresponding gain in the SSER can exceed three orders of magnitude. Alternatively, the shadow disc can be removed from the optical system and the fully reflective pick-off mirror42can be chosen to have a sufficiently large diameter (preferably at least 75% of the diameter of the lens43) to redirect a substantial amount of the scattered excitation beam away from the detector.

A preferred embodiment of the invention is henceforth disclosed, in which inexpensive stock optical components are used in the assembly of an optical system designed to measure 96-well microplates.FIGS. 5A and 5Billustrates the optical system and three-dimensional views of the excitation (FIG. 5A) and emission paths (FIG. 5B). The optical components are housed in a black anodized aluminum housing62, preferably made from multiple parts for ease of assembly.

A fiber optic bundle is inserted into the upper horizontal arm63, projecting a diverging excitation beam axially along the arm. The excitation light50emitted by the bundle has been obtained from either a laser or broadband optical source remotely from the inventive optical system using methods known in the prior art. The fiber optic bundles are preferably made from silica, with a numerical aperture of 0.22. The diameter of the bundle aperture is preferably 1 mm, with 19 fibers with a core diameter of 200 μm arranged in a hexagonal pattern.

A plano-convex lens51with a ½ inch diameter and a focal length of 20 mm is employed to collimate the excitation beam. Aperture52reduces the diameter of the collimated beam to 5 mm. Pick-off mirror53is a BK7 rod mirror with one side polished at an angle of 45° relative to the rod axis. The polished side is coated with aluminum to form a broadband mirror. The diameter of the rod is 10 mm. Such rod mirrors have recently become commercially available and are very inexpensive. The pick-off mirror53is attached to optical window55, which is preferably made of BK7 glass. The thickness of the window is 3 mm and the diameter is 30 mm. The pick-off mirror53directs the excitation beam upwards along the well axis, where it is intercepted by aspheric lens54. This lens is preferably an inexpensive stock molded-glass aspheric lens with a diameter of 30 mm and an effective focal length of 27 mm. The spatial offset between the reflective pick-of mirror53and the aspheric lens54is preferably less than 2 mm. The excitation beam is focused onto the optical well with a numerical aperture of 0.18. The excitation beam passes through the well5and its optical power upon transmission is directly measured with a large-area silicon detector61on the opposite side of the microplate well. The absorbance of the liquid sample6is measured in this fashion by referencing the measured optical power to the optical power without the microplate present in the conventional manner.

If the liquid sample is fluorescent, the emitted light is collimated by lens54, where it forms an axial beam that propagates towards the pick-off mirror53, as inFIG. 4B. Although the pick-off mirror attenuates the central portion of the emission beam, most of the power collected by lens54is retained. The beam passes through optical window55and is partially attenuated by shadow ring57. The shadow ring57is supported by rod56, which is attached to the bottom of optical window55. The diameter of the shadow ring is 21 mm, which shadows approximately 50% of the area of the initial collimated emission beam. The remaining ring of emission light is focused by lower aspheric lens58, which preferably is a stock molded glass aspheric lens with a diameter of 38 mm and a focal length of 30 mm. The emission light is then reflected by aluminized elliptical mirror59, which deflects the focusing beam again into the horizontal plane where it is focused onto the end face of fiber optic bundle60. The elliptical mirror59is preferably an inexpensive aluminized secondary mirror of the type used in Newtonian reflector telescopes. The emission bundle60is preferably an inexpensive borosilicate glass bundle with a numerical aperture of at least 0.4 and an end face diameter of 4 mm. The overall collection efficiency of the optical system is approximately 1%.

FIG. 6Aillustrates the performance of the optical system in terms of the rejection of scattered excitation light. The Figure displays all rays that retain more than 0.8% of the original power, which translates into all directly scattered rays. Rays originating from higher-order scattering processes are not shown. The cone of scattered excitation light71from the meniscus is clearly seen emanating from the microplate well5, whereupon the rays are collimated by the lens54. Again, the collimated rays slightly converge as they travel away from lens54. These rays are intercepted by shadow disc57and are prohibited from reaching the aperture of the emission bundle60. Although a group of rays72do succeed in bypassing the shadow disc, they are produced by scattering at the bottom side of lens54and are not imaged onto the emission bundle60. These rays therefore do not cause a decrease in the SSER.

The case of a decentered well is considered inFIG. 6B, in which the well axis and the optical system axis are offset by 0.4 mm. The offset causes the axial excitation rays to scatter from the meniscus with a slight angle, resulting in a tilt of the cone of scattered excitation rays75. The outermost rays within tilted cone are collimated at a position that is further from the optical system axis than inFIG. 6A. Fortunately, the diameter of the shadow disc57is sufficiently large to also intercept these rays. The optical system shown inFIG. 6Ais therefore robust and can tolerate lateral well misalignments of 0.4 mm, but preferably 0.25 mm.

The performance is further quantified inFIG. 7, in which the SSER is plotted as a function of the diameter of the shadow disc for the case of the preferred embodiment ofFIGS. 4 and 5. A shadow disc with a 10 mm diameter, which is equal to that of the pick-off mirror53, results in a low SSER of only 2.5. Note that if the pick-off mirror is replaced by a half-silvered mirror according to the prior art (shown inFIG. 1), then the SSER would be even lower. However, increasing the shadow disc diameter to 20 mm provides a dramatic increase in the SSER of three orders of magnitude. A diameter of 20 mm or more is also shown inFIG. 7to maintain a SSER of greater than 1000 even if the optical head is de-centered from the well axis by 0.25 mm.

The three order of magnitude increase in the SSER produced by the present invention enhances the signal-to-noise ratio of the optical system by a corresponding three orders of magnitude, provided that scattered excitation light is limiting the system detection limit. This will often be the case for measurements in which the absorption and emission bands of the fluorophore overlap or are spectrally adjacent. Alternatively, the present invention allows the user to choose simpler and less expensive optical filters with less rejection. This novel aspect is also accompanied by the dual measurement of absorbance and fluorescence in a single optical system, which alleviates the need for a second optical beam path for absorption measurements. Finally, it is important to note that as a result of the axial beam path through the optical system, the performance is insensitive to variations in sample volume (i.e. fluid height) and different meniscus radii of curvature.

Although the examples disclosed herein pertain to measurements of fluorescence through the bottom of a microplate well, the optical head may be mounted either above or below the microplate wells. In a preferred embodiment, the position of the optical head is user-configurable and may be moved to enable reading from below or from above the wells. If the optical head is mounted below the microplate wells, then either clear (i.e. polystyrene) microplates or clear-bottom black microplates may be used. Although clear-bottom black microplates are advantageous, the efficient rejection of scattered excitation light in the optical system and confocal nature of the fluorescence measurement provides excellent performance with clear microplates as well in many cases.

The optical system may be securely mounted with the microplate reader or assay instrument, and the reading of adjacent wells be achieved by moving the microplate on a two-dimensional translation stage. In this configuration, the optical source and optical detector, and accompanying filtering elements, may be directly integrated into the optical system using methods known in the prior art. Alternatively, the compact optical head may instead be moved relative to a stationary microplate. In this configuration, the filtered excitation light from a stationary light source subsystem is preferably delivered to the optical head via fiber a fiber optic bundle. The excitation delivery bundle may be bifurcated to provide an optical power reference beam. In addition, the emission beam is preferably delivered to a stationary filtering and detection subsystem via a fiber optic bundle. Furthermore, the absorbance detector61is preferably mounted on a separate mechanical arm that extends around the microplate and maintains stable alignment along the vertical axis of the optical system without causing mechanical interruption.

It will be apparent to those skilled in the art that there are many possible forms of the invention that, while differing from the aforementioned embodiments, do not depart from the scope and theme of the invention. For example, the pick-off mirror53may be replaced by a half-silvered mirror, and the transmitted beam can be used as a reference beam for monitoring the optical power of the excitation beam. Also, the pick-off mirror may be supported on a solid platform suspended in the center of the optical system rather than resting on an optical window55. Furthermore, the optical system may be adapted to the measurement of fluorescence and absorbance in 384-well microplates by decreasing the numerical aperture and/or focused spot size of the excitation beam.

Another modification of the above embodiments involves the use of metallic mirrors in the place of lenses. This approach has the threefold advantage of increasing the system bandwidth, reducing spurious reflections, and eliminating chromatic aberrations. Referring toFIG. 8, excitation light rays91from the light source90are focused onto the microplate well5, along the axis of the microplate well, by a first curved mirror94.

The optical power of the beam of excitation light is transmitted through the liquid sample is measured by a detector96positioned above the microplate well5. The fluorescent light98emitted from the fluorescent liquid6is collected, collimated, and redirected by a second concave mirror100. A hole102is cut through the second concave mirror100in order to allow the passage of the focused beam of light originating from the light source. The collimated fluorescent light104passes a shadow disc106, which absorbs excitation light scattered by the meniscus7. The ring of fluorescent light that remains after passing the shadow disc106is focused to a focal point110by an aspheric lens108.

In a variation of this embodiment, an on-axis parabolic mirror or off-axis parabolic mirror (not shown) may be used in place of the aspheric lens108to focus the collimated beam after it propagates past the shadow disc106. In a preferred embodiment, a molded glass aspheric lens108is used because of its low cost and because the final focusing section of the optical system is relatively immune to minor chromatic aberrations. Furthermore, since the position of the lens108is beyond the shadow disc106, the effect of scattering by the lens surfaces need not be considered. Finally, since the emitted light is red-shifted from the excitation wavelength, the transmission through a glass aspheric lens is most often sufficient, even if the excitation wavelength is in the ultraviolet part of the spectrum. This arrangement provides improved optical performance over the aforementioned preferred embodiment, at the expense of the additional cost of a low numerical aperture off-axis parabolic mirror.