DETAILED DESCRIPTION

[0085] FIG. 1 is a schematic diagram of one exemplary embodiment of a fluorescence spectrophotometer system constructed and operative in accordance with the present invention. A broadband light source 100 illuminates a mirror 102 , which is suitably curved to focus the light onto a double monochromator 104 . In alternative configurations, a narrow band light source such as a photodiode or a laser may be substituted for the broadband light source depicted in FIG. 1 ; those of skill in the art will appreciate that such a narrow band light source may be implemented in conjunction with the any or all of the fluorescence spectrophotometer systems and monochromator configurations set forth in detail below.

[0086] In some alternative embodiments, for example, light source 100 may comprise a halogen cycle tungsten filament lamp or other suitable lamp operative to transmit light to a spherical concave reflector system having interchangeable apertures; as described below, such a reflector system may be telecentric at both ends of the optical train and fully corrected for third order aberrations.

[0087] Light of the desired wavelength is passed by an excitation double monochromator 104 to a light transfer module (LTM) 106 . The LTM 106 directs the excitation light from monochromator 104 onto a sample 108 , which can be, for example, one well of a microwell plate or one lane of a 1-D polyacrylamide gel. Any resulting fluorescent or luminescent light emitted by the sample may be collected by LTM 106 as set forth below; LTM 106 may direct the light to the entrance aperture of an emission double monochromator 110 . In operation, monochromator 110 may be selectively adjusted to pass wavelengths from the emission spectrum of the fluorophore(s) in the sample, and is operative to direct those wavelengths to a photodetector 112 , which measures the energy of the emitted light.

[0088] Photodetector 112 may be any suitable photosensitive device, including but not limited to a photomultiplier tube (PMT), a phototransistor, or a photodiode. The electronic output of photodetector 112 may be applied to an automatic processing unit 114 , which generates a signal (which may be stored in a numerical form suitable for further analysis) indicating detection of the selected emission. Automatic processing unit 114 may be, for example, a personal computer having a data collection interface to the spectrophotometer system In general, all of the elements of the optical pathways in the instrument depicted in FIG. 1 , including double monochromators 104 and 110 and the LTM 106 , may be isolated in light-tight boxes coated internally with non-fluorescent absorptive material to minimize reflectance and light from other sources such as room light.

[0089] FIG. 2A is a schematic diagram of an embodiment of a double monochromator configured and operative for use in embodiments of a fluorescence spectrophotometer system The illustrated configuration may be used for either the excitation double monochromator 104 or the emission double monochromator 110 illustrated in FIG. 1 .

[0090] Broadband light is introduced through an entrance aperture 200 in the double monochromator and reflects off the front surface of a first holographic concave grating 202 that is pivotable about an axis 203 . Use of front surface reflection enhances the efficiency of a double monochromator by avoiding absorption of light within an optical support structure, such as in a rear surface reflection glass mirror. Use of a concave grating allows incident light to be dispersed into selectable wavelengths without the use of supplemental collimating mirrors. This design makes it possible to eliminate two collimating mirrors per grating used in conventional dual monochromator instruments. Use of a holographic grating also reduces astigmatic aberrations, and thus decreases the amount of light of unwanted wavelengths (“stray light”).

[0091] Each double monoclromator has three apertures: an entrance aperture 200 , through which light first enters the monochromator; an internal (or “first”) selection aperture 206 ; and an exit (or “second”) selection aperture 216 , through which light exits the monochromator. The first concave grating 202 reflects wavelengths of the incoming light as a first spatially dispersed beam 204 . Each wavelength of this first spatially dispersed beam 204 is reflected at a unique angle relative to other wavelengths. Pivoting first concave grating 202 about its axis 203 enables a selected band or range of wavelengths 208 to be directed through the internal selection aperture 206 within the monochromator housing.

[0092] The selected range of wavelengths 208 is then reflected off a second holographic concave grating 210 that is pivotable about an axis 211 . Second concave grating 210 reflects the selected range of wavelengths 208 of the first spatially dispersed beam 204 as a second spatially dispersed beam 212 . Pivoting second concave grating 210 about its axis 211 enables a selected narrow band of wavelengths 214 to be directed through exit selection aperture 216 .

[0093] The dimensions of selection apertures 206 and 216 determine the selected wavelengths of light leaving the monochromator. Wider apertures allow more light energy to pass through a monochromator, but the light includes a broad range of wavelengths; narrower apertures reduce the amount of light passing through a monochromator but narrow the selected range of wavelengths. The use of wider apertures increases the sensitivity of detection, which may be beneficial in measurements of total fluorescence in an area or volume (e.g. measurements made in microwells). In contrast, the use of narrow apertures increases the spatial resolution of a monochromator, which may be beneficial in discriminating between different fluorophores at particular locations (e.g. measurements of bands separated in a polyacrylamide gel). That is, the smaller the aperture dimensions, the smaller the area of detection at the sample. In the case of laser light sources and pinhole apertures, for example, the area of excitation at any given moment is a point that corresponds to a particular data pair representing fluorescent intensity and position.

[0094] It is generally known by those of skill in the art of optical systems that each optical component reduces the efficiency of light throughput. Advantages of the configuration shown in FIG. 2A include, but are not limited to, elimination of collimating mirrors for redirecting light within the double monoclromator, such as is the case with traditional monochromators. In the exemplary embodiment, only two light directing elements (the first and second holographic concave gratings 202 , 210 ) are required, thus improving overall light efficiency.

[0095] In the configuration illustrated in FIG. 2 A, the first concave grating 202 and the second concave grating 210 may be pivoted oppositely and in tandem by a suitable mechanism. A mechanism according to one embodiment (illustrated in FIG. 2B ) implements a tension band actuator mechanism for pivoting gratings 202 , 210 . In the FIG. 2B configuration, gratings 202 , 210 are mounted coaxially with pivot wheels 250 , 252 , respectively. One end of a lever arm 254 is connected to pivot wheel 250 ; the other end of lever arm 254 is coupled to a screw drive mechanism 256 . Lever arm 254 traverses along threaded rod 258 as the rod 258 is rotated by a motor 260 .

[0096] Lever arm 254 movement along rod 258 causes pivot wheel 250 to rotate. Pivot wheel 250 is connected to pivot wheel 252 by a tension band 262 . In accordance with the FIG. 2B embodiment, tension band 262 may be a 0 . 003 inch stainless steel band. Tension band 262 causes pivot wheel 252 and grating 210 to counter-rotate synchronously and in tandem with pivot wheel 250 and grating 202 . A spring 264 connected between a housing (not shown) and pivot wheel 252 maintains tension in tension band 262 . The position (i.e. angular orientation) of gratings 202 , 210 may be determined and controlled by a microcontroller connected to pivot wheel sensor 266 and screw drive sensor 268 .

[0097] The FIG. 2B embodiment is provided by way of example only, and not by way of limitation; it will be appreciated that alternative mechanisms exist for pivoting gratings 202 , 210 either synchronously or otherwise. For example, each grating 202 , 210 may be provided with a respective pivot mechanism such that the angular orientation of gratings 202 , 210 relative to each other may be independently selectable. Additionally, alternative means for coupling gratings 202 , 210 for synchronous and complementary rotation are within the scope and contemplation of the present disclosure.

[0098] FIG. 3 is a schematic diagram of one embodiment of the LTM 106 shown in FIG. 1 . Excitation light enters LTM 106 through an entrance aperture whereupon it is directed, either with or without one or more mirrors 300 , to a coaxial excitation mirror 302 . The coaxial excitation mirror 302 may be flat, or include appropriate curvature to focus or to disperse light as desired. Coaxial excitation mirror 302 is positioned to direct the excitation light directly onto the sample 108 . More particularly, coaxial excitation mirror 302 may be positioned somewhat off-axis with respect to sample 108 , but should be positioned so that substantially all of the excitation light strikes the sample to achieve maximum illumination.

[0099] In one application, each well of a multi-well plate can be positioned beneath the coaxial excitation mirror 302 by X-Y translation of either LTM 106 , the multi-well plate, or both. In another application involving monochromators equipped with optional microscope optics, different regions of intact biological cells that have been mounted on glass slides or culture plates can be separately imaged using optical elements in the light path and by positioning the sample beneath coaxial excitation mirror 302 by X-Y-Z translation of either LTM 106 , the glass slides or culture plates, or both. Fluorescence emission from one or more fluorophores in a sample may be collected by a coaxial emission mirror 304 . Coaxial emission mirror 304 may be concave so as to focus and direct the emission light, either directly or by one or more light directing mirrors 306 , out of an exit port or aperture of LTM 106 . In the embodiment shown in FIG. 3 , emission light exits LTM 106 from the same side that excitation light enters LTM 106 . However, different placements of the entrance and exit apertures may be used by suitable placement of light directing mirrors 300 , 306 .

[0100] In this context, “coaxial” generally refers to the position and orientation of mirrors 302 , 304 relative to an area to be illuminated (in the case of excitation mirror 302 ) and relative an area that is emitting fluorescent or luminescent light (in the case of emission mirror 304 ). The coaxial placement of excitation mirror 302 with an area to be illuminated, as well as the coaxial placement of emission mirror 304 with an area emitting light, combine to ensure that a high percentage of excitation light is directed onto the sample within a well 108 , for example, and that a high percentage of the fluorescent or luminescent light emitted from the opening of well 108 is collected for analysis. In some embodiments, emission mirror 304 may be positioned slightly off-axis relative to excitation mirror 302 to avoid interference in the optical train.

[0101] In a particularly efficient embodiment, all of the mirrors within LTM 106 comprise front, or “first,” surface mirrors. First surface mirrors have a reflective material, such as aluminum or other reflective metal, for example, coated onto the surface of a substrate, such as glass or ceramic; incident light is directed onto the reflective coating. Since the coating serves as the reflective surface, incident light does not penetrate the substrate as in an ordinary second surface mirror. Accordingly, such first surface mirrors are substantially more efficient than traditional second surface mirrors.

[0102] It will be understood by those of ordinary skill in the art that a number of different reflecting mirrors may be used to direct light within LTM 106 , as needed. As noted above, however, it is generally desirable to minimize the number of such reflecting surfaces in order to improve efficiency of LTM 106 . In the FIG. 3 embodiment, excitation mirror 302 may be an elliptical mirror approximately 6×9 millimeters in dimension, while the emission mirror 304 may be approximately 75 millimeters in diameter. Other dimensions can be used and generally will vary with the dimensions of the overall instrument.

[0103] FIG. 4 is a schematic diagram of a simplified embodiment of a fluorescence spectrophotometer. As in the embodiment described above with reference to FIG. 1 , excitation wavelengths may be selected from a broadband light source 100 by means of an excitation double monochromator 104 . The excitation light is directed by a coaxial excitation mirror 302 to a sample within a well 108 . Fluorescent and luminescent emission light may be collected and focused by a coaxial emission mirror 304 that is in reflective alignment with the coaxial excitation mirror 302 . Emission mirror 304 directs the collected and focused light into an emission double monochromator 110 . As described above, monochromator 110 directs one or more selected wavelengths of emission light to a photodetector and analyzer module 112 for counting and analysis. This embodiment minimizes the number of reflective surfaces within LTM 106 .

[0104] The compact configuration of the embodiment shown in FIG. 4 allows for use of the instrument as a dual path spectrophotometer. That is, a sample can be illuminated with excitation light either from the open side of well 108 or from the bottom side of well 108 as set forth below.

[0105] FIG. 5A shows a first alternative dual path embodiment of a fluorescence spectrophotometer system One or both of the light source 100 and the excitation double monochromator 104 optionally may be translated from a first position (illustrated in FIG. 5A by dashed lines) to a position below a multi-well plate such that excitation light impinges upon a bottom-illumination coaxial excitation mirror 302 ′, which directs excitation light through the transparent bottom substrate of a well 108 . Alternatively, if light source 100 is not translated, light directing mirrors may be interposed to direct light through excitation double monochromator 104 . Fluorescence emissions emanating from the opening of well 108 may be collected by emission mirror 304 . When the system is configured for bottom-illumination, the top-illumination excitation mirror 302 (illustrated in FIG. 4 , but not shown in FIG. 5A ) may be removed from the light path in order to maximize the amount of fluorescent and luminescent light collected by the emission mirror 304 . Alternatively, top-illumination excitation mirror 302 can be left in place. In either case, bottom-illumination excitation mirror 302 ′ is in direct alignment (as opposed to reflective alignment) with emissions mirror 304 .

[0106] FIG. 5B shows a second alternative dual path embodiment of a fluorescence spectrophotometer system In this configuration implemented for bottom illumination, a set of one or more redirection mirrors 303 A, 303 B may be interposed into the light path from excitation double monochromator 104 in order to intercept and to direct excitation light. The intercepted excitation light is redirected to impinge upon a bottom-illumination coaxial excitation mirror 302 ′, which directs the excitation light through the transparent bottom substrate of well 108 . The top-illumination coaxial excitation mirror 302 may be left in place, as shown, or moved out of position when the redirection mirrors 303 A, 303 B are moved into position. Such movement may be accomplished, for example, by translation (e.g. along an axis into or out of the page) of a carriage on which all four mirrors 302 , 303 A, 303 B, 302 ′, are mounted. As with the FIG. 5A embodiment, bottom-illumination excitation mirror 302 ′ is in direct alignment (as opposed to reflective alignment) with emission mirror 304 .

[0107] In all of the configurations shown in FIGS. 4, 5A , and 5 B, additional redirection mirrors may be used as required to guide light to selected locations within the instrument.

[0108] According to an embodiment, a timer-counter board that operates at a frequency in excess of 100 MHz may be used. The photodetector and analyzer module (designated by reference numeral 112 in FIGS. 4, 5A , and 5 B) may be configured to respond to photons at frequencies as high as 30 MHz. The dynamic range of this system ranges from 0 to more than 30×10 ⁶ photons, thereby eliminating the dynamic range limitations which previously restricted the use of photon counting in fluorescence detection. If the observed current from the PMT is greater than a prescribed threshold established at the photodetector, a 5 volt electrical pulse having a duration of approximately 2 nanoseconds is produced; the timer-counter circuit enumerates these pulses as a function of time, that is, establishes a direct quantitation for photons per unit time.

[0109] Using a spectrophotometer according to one embodiment, a pre-cast polyacrylamide gel containing fluorescently labeled nucleic acids or proteins was placed on a flat plate in the positioning mechanism directly under the LTM. With the excitation and emission monochromators set at wavelengths suitable for the fluorescent labels, the gel was translated (in the X and Y directions) under the LTM until all of the gel area had been traversed. At each point of this travel, a fluorescent reading was made and stored as a two dimensional array representing the fluorescent emission at each point of the gel. The distance between the points was adjusted to yield the best response for a given data acquisition time. In the actual experiments, the gel was also scanned as “lanes” representing the path of electrophoresis from the sample well at the top of the gel to the base of the gel because the fluorophores in an electrophoresis gel are arrayed in a line rather than as a point. Each such lane was scanned from the sample well to the bottom of the gel to achieve a substantial reduction in overall data collection time. As a reference for background, a blank lane was scanned and the data subtracted on a point-by-point basis from the corresponding data for lanes containing fluorophores. The corrected data were then analyzed in two ways. In one analysis, an image was constructed of the original gel which was compared to standard laboratory photographs of the same gel for evaluation of standard gel parameters such as migration distance, separation of molecules, and concentration of molecular species as determined separately by the digital image and the film. In the other analysis, a “densitometry” plot equivalent to those made for gel lanes from autoradiography films using flat bed scanning detectors was created. From the database relating fluorescence intensity of a lane, the center of each fluorescent band was identified and a cross sectional graph of fluorescent intensities as a function of migration distance was prepared. From the use of gels prepared with different but known amounts of the same fluorescent labeled nucleic acids, a standard curve establishing lower and upper limits of sensitivity, resolution, and overall dynamic range for gel detection were determined.

[0110] The “square intensity point spread function” and the “long penetration depth” properties of one and two photon absorption processes have been recognized as important features in future developments in fluorescence detection. Both are accomplished by focusing a femtosecond short pulse laser onto a focal plane in a sample to be studied for fluorescence. In a spectrophotometer according to another embodiment, a laser beam from an appropriate laser was substituted for the quartz halogen or xenon light source used for excitation. The laser beam was used to illuminate the entrance aperture of the excitation monochromator. Additional modifications where needed in some cases including, for example, the use of one or two pinhole apertures rather than the standard rectangular slits, and the insertion of an objective lens to focus the light after it had passed through the monochromator. The fluorescence emission was collected through the light transfer module as previously and the fluorescence measured as a function of time, or in the cases of image formation and area scanning, as a function of time and position of the light transfer module over the sample as described in the gel analysis above. In this configuration, a pinhole exit aperture on the excitation monochromator was used to image light onto a sample, and each point of the image used to excite fluorescence. Moving the sample position in an x-y-z fashion enabled scanning of areas of the sample to create an image or database. For confocal microscopy, two pinhole apertures were required as described below. For multi-photon applications, only a single pinhole aperture was used as the exit aperture of the excitation monochromator. For two-photon excitation, a microlens array could be used if needed to focus the beam for high transmittance. In general, the configuration was epi-fluorescent although in certain polarization applications, excitation was from the bottom and emission light was collected from the top.

[0111] In yet another embodiment, a fluorescence spectrophotometer system constructed and operative in accordance with the present invention was applied in the creation of a scanning fluorescence polarization detector. Fluorescent molecules in solution, when excited with plane polarized light, will emit light back into a fixed plane (i.e. the light remains polarized) if the molecules remain stationary during the fluorophore's period of excitation (excited state lifetime). Molecules in solution, however, tumble and rotate randonly, and if the rotation occurs during the excited state lifetime and before emission occurs, the planes into which light is emitted can be very different from the plane of the light used for the original excitation.

[0112] The polarization value of a molecule is proportional to its rotational relaxation time, which by convention is defined as the time required for a molecule to rotate through an angle of 68.5°. Rotational relaxation time is related to solution viscosity (η), absolute temperature (T), molecular volume (V), and the gas constant (R):

Polarization value∝Rotational Relaxation Time=3ηV/RT

[0113] If viscosity and temperature are constant, the polarization is directly related to molecular volume (molecular size), which, in general, correlates well with molecular weight. Changes in molecular volume result from several causes, including degradation, denaturation, conformational changes, or the binding or dissociation of two molecules. Any of these changes can be detected as a function of changes in the polarization value of a solution. Specifically, a small fluorescent molecule which rotates freely in solution during its excited state lifetime can emit light in very different planes from that of the incident light. If that same small fluorophore binds to a larger molecule, the rotational velocity of the small molecule decreases and the effect is detected as a decrease in the polarization value. Measurement of the effect requires excitation by polarized light which can be obtained using a laser or through light selection using a polarizing filter which only transmits light traveling in a single plane. In one experiment, light of a defined wavelength obtained from one embodiment of an excitation monochromator operative in accordance with the present invention was further refined by passing the light through a polarizing filter (designated the “polarizer”) to obtain monochromatic, plane polarized light for excitation (for the present purposes designated “vertically polarized light”). Concurrently, the light path for collecting the emission light was similarly modified by selective introduction of one of a pair of polarizing filters (designated the “analyzers”), one of which can be rotated to a position vertical to the plane of the excitation light, whereas the other of which can be rotated to a position horizontal to the plane of the excitation light. When a fluorescent sample in solution was introduced into the light path between the polarizer and the analyzers, only those molecules which were oriented properly to the vertically polarized plane absorbed light, became excited, and subsequently emitted light. By selecting the appropriate analyzing filter to be inserted into the emission light path, the amount of emitted light in the vertical and horizontal planes can be measured and used to assess the extent of rotation of the small fluorescent molecule in the solution before and after binding to a larger molecule. FIG. 6 , described below, illustrates a system which may facilitate the forgoing experiment.

[0114] In yet another application, a fluorescence spectrophotometer system was utilized in confocal microscopy—a method for eliminating one of the fundamental difficulties of fluorescence microscopy, namely, the reduction in spatial resolution at the focal plane of the microscope owing to out-of-focus light. A spectrophotometer according to another embodiment was used to create a confocal microscope from the embodiment essentially as described under laser excitation above to focus a light image on whole cell mounts and to achieve both multiple and single photon excitation of the fluorescent labels in a sample. Fluorophores in planes out of the focus were not illuminated and did not fluoresce.

[0115] In confocal imaging, apertures were used in both the excitation and emission light paths in order to focus a cone of light through the specimen and in the emission light path in order to eliminate scattered and out-of-plane fluorescence. The development of mode locked dye lasers has made simultaneous multi-photon excitation practical because such lasers are capable of delivering the available excitation energy to a focal spot in very brief pulses and with sufficient energy to achieve two photon excitation. In multi-photon imaging, the focal spot provided by the laser excites a sufficiently small volume that, when used in conjunction with the light transmission module described above, makes it possible to collect all emission light without a second pinhole aperture on the emission side. No emission aperture changes were necessary.

[0116] FIG. 6 is a schematic diagram of an embodiment of a fluorescence spectrophotometer system including fluorescence polarization as noted briefly above. In the FIG. 6 embodiment, light exiting the excitation monochromator 104 may be plane polarized In that regard, an excitation polarizing filter 602 may be operative to polarize the excitation light in a selected orientation. The polarized excitation light is transferred to the sample well 108 via LTM 106 . Resulting fluorescent and luminescent light emitted by the sample is collected by LTM 106 , which directs emission light to a polarization analyzer 604 . In operation, polarization analyzer 604 may determine if the light emitted from well 108 has been rotated from its original orientation 602 . Such rotation may provide information about the sample in well 108 , for example, the spin and/or size of molecules in the sample. Analyzer 604 transmits the emission light to the entrance aperture of emission double monochromator 110 as described above with reference to the embodiment shown in FIG. 1 .

[0117] Excitation polarizing filter 602 may generally be implemented as an optical filter operative to restrict the excitation light to plane polarized excitation light oriented in a selected plane, as is generally known in the art. An optical filter holder (not shown) may be employed selectively to insert the optical polarizing filter 602 into the path of the excitation light.

[0118] FIG. 7 is a simplified diagrammatic view of one embodiment of a polarization analyzer 700 constructed and operative in accordance with one embodiment of the present invention. Analyzer 700 generally corresponds, to and incorporates all of the functionality of, analyzer 604 described above with reference to FIG. 6 . Analyzer 700 may include an aperture 702 operative to admit emission light (from LTM 106 as indicated in FIG. 6 ) and two emission polarizing filters 704 and 706 , which may be disposed on a filter holder such as plate 708 , for example. A first emission polarizing filter 704 has a polarization parallel to that of the excitation polarization filter 602 , and hence passes light having the original orientation produced by the excitation polarizing filter 602 . A second emission polarizing filter 706 , on the other hand, has a polarization perpendicular to that of excitation polarizing filter 602 , and hence blocks light having the original orientation. A motorized slider 710 or other suitable mechanism may slide filter plate 708 between two positions: a first position in which the first (parallel) polarization filter 704 is aligned with aperture 702 in the light train, and a second position in which the second (perpendicular) polarization filter 706 is aligned with aperture 702 in the light train.

[0119] Two measurements may be taken for each sample. For the first measurement, the motorized slider 710 slides the filter plate 708 into the first position and any emission fluorescence from the sample may be passed through the parallel filter 704 . For the second measurement, the motorized slider 710 slides the filter plate into the second position, and any emission fluorescence from the sample may be passed through the perpendicular filter 706 . The amount of rotation, if any, may be determined by comparing the two measurements. For example, if the polarization of the excitation light has not been rotated in the sample, no emission fluorescence should be detected in the second measurement, since all of the light in the original orientation would be blocked by the perpendicular filter 706 .

[0120] Though polarization analyzer 700 has been illustrated and described as a discrete component (e.g. reference numeral 604 in FIG. 6 ) of a fluorescence spectrophotometer system, it will be appreciated that the components detailed in FIG. 7 or their equivalents, as well as the polarization analyzer functionality, may alternatively be incorporated into the emission side of LTM 106 or into monochromator 110 illustrated in FIG. 6 . As set forth in detail below, some embodiments incorporate emission polarization analyzer functionality in the LTM upstream of the emission mirror in the light train. Similarly, an excitation polarizing filter (reference numeral 602 in FIG. 6 ) may be incorporated into monochromator 104 or the excitation side of LTM 106 .

[0121] FIG. 8 is a simplified cross-sectional diagram of an illumination system Illumination system 800 may generally correspond to light source 100 illustrated in FIGS. 4, 5A , and 5 B. Illumination system 800 may be operative to image the filament of a light source or lamp 801 onto the entrance aperture 200 of an excitation monochromator such as represented by reference numeral 104 in FIG. 1 . In the FIG. 8 embodiment, a first spherical reflection surface 802 and a second spherical reflection surface 803 cooperate to form a spherical concave reflector system, in this case an Offner 1:1 afocal relay. Spherical surfaces 802 , 803 may be used to correct for all third order aberrations; the spherical concave reflector system is telecentric at both ends of the optical train.

[0122] As is generally known in the art, lamp 801 may include a rear mirror 804 operative to reflect the flux from the rear of the filament forward through the system. Additionally or alternatively, an aperture wheel having interchangeable apertures (not shown) may be inserted into the light path. Rotation of the aperture wheel enables selection of one of a plurality of apertures to be inserted into the light path illustrated in FIG. 8 , for example; accordingly, the cone angles of the flux incident on entrance aperture 200 may be selectively adjusted in accordance with system requirements.

[0123] In that regard, means for selectively inserting one of a plurality of apertures into the path of light moving through the spherical concave reflector system of illumination source 800 may be included. Mechanisms such as linear actuators, gears for rotating an aperture wheel, and the like are contemplated. Various methods of interposing one or more apertures into an optical train are known in the art.

[0124] FIG. 9 is a simplified cross-sectional diagram of one embodiment of a light transfer module incorporating a compound parabolic concentrator. As indicated by the arrow in FIG. 9 , excitation light may be admitted to LTM 106 (generally represented by the dashed lines in FIG. 9 ) from the left side of the diagram, and is directed through an aperture 902 by excitation mirror 302 (see FIGS. 4, 5A , and 5 B), which is appropriately mounted as shown, to illuminate a sample. A compound parabolic concentrator (CPC) 901 may receive emission light (i.e. fluorescent or luminescent light emitted from the illuminated area containing sample) through aperture 902 .

[0125] In accordance with this embodiment, CPC 901 may comprise a polished, reflective surface operative to collect the flux radiated from the illuminated sample and to concentrate emission light for reflection by an emission mirror (not shown in FIG. 9 ) such as described in detail above.

[0126] As noted above, the various components of the emission polarization analyzer (see FIGS. 6 and 7 ) may be interposed between CPC 901 and the emission mirror, incorporating the functionality of a polarization analyzer into LTM 106 . This arrangement may substantially reduce the influence of the LTM 106 itself on the polarization state of the emission light.

[0127] FIG. 10A is a simplified cross-sectional diagram of the interior of a light transfer module incorporating a compound parabolic concentrator, and FIG. 10B is a simplified cross-sectional diagram of one embodiment of a light transfer module incorporating emission polarization upstream of an emission mirror. The views depicted in FIGS. 10A and 10B are parallel to the light train and are taken at lines 10 A and 10 B, respectively, shown in FIG. 9 ; i.e. emission light directed through aperture 902 of CPC 901 in FIG. 10A passes through the polarizer unit 910 in FIG. 10B before being directed by an emission mirror to an emission double monochromator as described above.

[0128] In the FIG. 10A embodiment, an aperture 903 admits excitation light from an excitation double monochromator into LTM 106 as described above with reference to FIG. 9 . As noted above, emission light emitted by the illuminated sample passes through aperture 902 and is directed by the polished interior surface of CPC 901 to an emission mirror.

[0129] Polarizer unit 910 illustrated in FIG. 10B may be interposed between CPC 901 and an emission mirror, such that emission light may be polarized or otherwise filtered before it impinges on the emission mirror. In operation, an actuator or drive motor 907 , for example, coupled to a filter holder or wheel 904 may enable selection of one of a plurality of polarizing filters (represented by reference numerals 905 A and 905 B) or other filters (represented by reference numeral 906 ). One or more slots (represented by reference numeral 908 ) in wheel 904 may be provided with no filter at all. Accordingly, a filter holder such as wheel 904 may be selectively operative to insert one of a plurality of polarizing or other filters into the path of the emission light. The quantity, nature, and polarization plane of the various filters incorporated in wheel 904 may be selected as a function of overall system requirements.

[0130] It will be appreciated that alternative or additional mechanisms may be implemented to enable selection of filters in polarizer unit 910 . Linear translation of a filter plate such as described above with reference to FIG. 7 , for example, may be appropriate and equally effective, depending upon size and operational requirements of the LTM in the context of the overall spectrophotometer system.

[0131] It will also be appreciated that the foregoing descriptions of the drawing figures are exemplary only, and that the disclosed embodiments are susceptible to modifications and alterations which may improve overall system efficiency. For example, rotating aperture wheels and appropriate mountings may additionally be implemented in the LTM 106 of FIG. 9 ; apertures may be selected in accordance with the dimensions of a sample well, for instance, to adjust excitation light passed through aperture 902 such that stray light may be minimized.

[0132] In one embodiment of a double monochromator such as depicted in FIG. 2 A, for example, baffles may be incorporated to reduce stray light originating from diffracted flux which does not pass through the first selection aperture 206 . Spectroradiometer measurements of monochromator designs without baffles have revealed that considerable flux which is half the desired wavelength may be transmitted through the first selection aperture; for example, if a conventional excitation monochromator is set to pass light having a wavelength of 600 nm, then flux having a wavelength of 300 nm may also be transmitted through the selection aperture as second order aberrations. Accordingly, reduction of stray light through implementation of baffles may represent an important determinant with respect to the purity of the light output by the monochromator.

[0133] FIG. 11 is a simplified flow diagram illustrating one embodiment of a method of analyzing a sample. As set forth in detail above with reference to the system embodiments, a method of analyzing a sample generally comprises providing excitation light from a light source (block 1101 ), directing the excitation light through a first double monochromator (block 1102 ), transmitting the excitation light to the sample through a light transfer module(block 1103 ), employing the ligh