WO2010111166A2

WO2010111166A2 - Compact dual pass monochromator

Info

Publication number: WO2010111166A2
Application number: PCT/US2010/028104
Authority: WO
Inventors: Josef Atzler; Andreas Kenda
Original assignee: Beckman Coulter, Inc.
Priority date: 2009-03-23
Filing date: 2010-03-22
Publication date: 2010-09-30
Also published as: WO2010111166A3

Abstract

A dual pass monochromator for generating excitation radiation and isolating emission radiation at prescribed wavelengths, useful for analyzing florescence in multi-assay micro-titer plate readers is disclosed. The optically dispersive element can be used to receive radiation through an entrance aperture; isolate a prescribed wavelength band; and then direct the prescribed wavelength band through a first exit aperture onto a sample. The excited emissions from the sample can then be received back through the first exit aperture and be directed to the optically dispersive element to isolate the emission wavelength band and direct it onto a detector through a second exit aperture. Band pass elements can be optically coupled to the optically dispersive element to tune the excitation and emission wavelength bands. Band pass optical elements can be dispersive diffraction gratings, or non-dispersive optical filters. The dual pass monochromator can be modular and include a number of optically isolated compartments.

Description

COMPACT DUAL PASS MONOCHROMATOR

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a non-provisional of and claims the benefit of U.S. Patent Application No. 61,210,865, filed on March 23, 2009, which is herein incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Embodiments of the present invention relate to a compact all-in-one monochromator device for illuminating fluorescing samples at a first wavelength and reading the emitted fluorescence signal at a longer second wavelength. An embodiment of the invention, which is particularly useful for use in multi-assay micro-titer plate readers, allows for building of a highly compact, integrated monochromator for the selection of both the excitation and emission wavelengths using a minimized number of optical components. Embodiments of the invention also allow for simple confocal sample interfacing.

[0003] In life sciences, samples are routinely characterized by examining their fluorescence properties. Typically in such studies, the actual sample, e.g. cells, tissue, proteins, DNA strings, etc., are treated with fluorophore-labelled molecular markers. The treated sample is then irradiated with light of a first wavelength band, the excitation wavelength, which causes the fluorophores present in the sample to emit fluorescence. From the emitted fluorescence, a second wavelength band, the emission wavelength, is selected and measured. Recording this emitted radiation allows one to identify and, within certain limits, quantify the fluorophores present in the sample and thus identify the structures and/or substances present.

[0004] Measuring said fluorescence signals requires first and foremost an optical system that reliably differentiates between excitation and emission radiation. Reliable fluorescence measurements with good signal-to-noise ratios require a blocking factor of at least 10⁴:l (preferably 10⁶:l or higher) between (scattered) excitation radiation and the fluorescence signals to be measured. Second, the instrument should be flexibly applicable, i.e. the device has to be able to measure at different excitation / emission wavelength combinations over as wide a spectral range as possible. A third important parameter is the collection efficiency. To detect even weak fluorescence signals, the light emitted by the sample should be collected over a wide solid angle and transferred to the detector with as little loss as possible. Finally, with a clear technological trend pointing towards integrated multi-purpose readers, it would be a significant technological and economic advantage to dispose of a compact modular monochromator device that could be inserted into a modular reader platform.

[0005] With an ever growing market for rapid substance screening, several attempts to solve these problems have been previously reported.

[0006] The problem of differentiating between excitation and emission signals with a high blocking factor is routinely addressed using i) non-dispersive optics, i.e. some type of filter, ii) dispersive optics, i.e. gratings or prisms, in combination with entrance and exit apertures, or iii) combinations of these principles.

[0007] The classical non-dispersive arrangement uses one bandpass filter for selecting a specific excitation wavelength from a broadband light source and a second bandpass filter for selecting a specific, non-overlapping emission wavelength from the emitted light. While using modern interference bandpass filters allows for blocking ratios in excess of 10⁵ : 1 , the use of such filters can reduce the overall flexibility of the system. An excitation/emission filter combination can only be used for specific fluorescent dyes. While the method of choice for most imaging applications, e.g. in microscopy, this presents a critical limitation for multipurpose fluorescence reading. Some instruments counter this problem by providing a number of different bandpass filters on filter wheels, typically one for excitation wavelength selection and one for filtering the emission, as outlined e.g. in US 6187267 Bl. Still, the number of different dyes to be analyzed is limited by the number of filters present in the system. Similar restrictions apply when using (monochromatic) laser sources for excitation. Also, the presence of filter wheels, or of multiple laser sources, makes such devices comparatively large.

[0008] Alternative filter instruments use tunable or graded optical filters. For example, DE 102004049770 Al / US 20060077383 Al disclose a system for measuring fluorescence using linearly variable filters. Using two such filters, would - in theory - allow one to continuously select any excitation/emission wavelength combination. However, such designs suffer from limited bandwidths, the inability to measure spectrally close excitation and emission wavelengths, and relatively poor spectral blocking ratios when operated at high throughputs.

[0009] Most commercial fluorescence assay readers use dispersive monochromators based on diffractive reflection gratings. Typically, radiation emitted by a broadband light source passes through a first monochromator selecting the required excitation wavelength. This excitation radiation is then directed onto the sample. The emitted fluorescence is collected and passed through a second monochromator that selects the emission wavelength of interest from the collected radiation and directs it onto a suitable detector. To improve blocking ratios, contemporary fluorescence detection systems use double grating monochromators for both the excitation and detection monochromators to better separate the wavelengths of interest from undesired spectral components. By tuning the grating component(s), such monochromators can easily be adjusted to specific wavelengths. Many layouts using such arrangements have been disclosed, e.g. EP 0540966 Bl, EP 0981043 Al, WO 0063680 Al or US 6187267 Bl, and/or are in practical use, like Tecan's Quad4 system. The common limitation is that using two separate double grating monochromators makes these instruments relatively large, which is typical when using two separated beam paths for excitation and emission radiation.

[0010] In addition to filter-only solutions and dispersive-only solutions, a number of dispersive / non-dispersive hybrids have been described, e.g. in EP 0981043 Al, WO 0063680 Al, US 6187267 Bl and US 6278521 Bl. The hybrid solutions are also relatively large and suffer from many of the same flexibility limitations as the filter-only solutions.

[0011] Another related problem involves the interface between the fluorescence analyzer and the sample, (i.e. how to effectively transfer the excitation light to the sample and the emitted fluorescence from the sample to the emission analyzer). When working with cuvettes, usually an angular setup is used in which the excitation and emission beam paths are angularly displaced from one another to minimize collecting stray radiation with the fluorescence signal. With the micro-titer plates used in modern multi-assay screening, such an arrangement is usually not feasible because the fluorescence signals of the individual sample wells have to be read either from above or through their bottoms.

[0012] A first work-around, prevalently used in fluorescence microscopy, is to use confocal excitation/emission optics in combination with a dichroic beam splitter separating the fluorescence from shorter-wavelength excitation light. The main set-back of this concept is that adding a dichroic component introduces an additional spectral characteristics into the system. To be effective, the spectral cutoff of the dichroic mirror must be between the excitation and the emission wavelengths. When analyzing different fluorescent dyes it thus may be necessary to switch the dichroic mirror between readings. Additionally, the extra element also reduces the light throughput and thus the instrument's sensitivity is also reduced.

[0013] To avoid these limitations, various optical systems have been designed to look at the sample well from the same side with separate excitation and emission beam paths. EP 0981043 Al discloses an arrangement where well focused excitation light is projected onto the sample well through a hole in the collimation mirror used to collect the (undirected) fluorescence radiation. WO 0063680 Al suggests an essentially similar system, the difference being that the excitation radiation is injected via a small plane mirror located between the collection mirror and the sample. While using a collimation mirror with high numerical apertures allows collecting fluorescence light over a wide solid angle, the drawback of both systems is the need for a well collimated excitation beam (preferably a laser), and the inevitable light losses caused by a hole in the collection mirror or the presence of an in-coupling mirror in the collection beam path. An alternative disclosed in US 6236456 Bl uses angularly separated excitation and emission beam paths to excite and detect fluorescence through the bottom of a sample well. The advantage of having optically decoupled, non-interfering beam paths is, however, diminished by the inherent limitation of the numerical apertures of the optics used, and hence a lower collection efficiency.

[0014] Between them, the aforementioned principles teach how to measure the fluorescence of fluorescent substances using two separate beam paths for excitation and emission. While powerful and flexible in use, the need for two separate spectral analyzers limits the degree of miniaturization possible and introduces the need for using sub-optimal workarounds for analyzer - sample interfaces, at least when measuring micro-titer plates.

[0015] Embodiments of the invention disclosed herein offer a way to overcome these obstacles and provide for a compact, simple and cost-effective device capable of measuring samples, in particular in micro-titer plates, with a minimal number of optical components and a confocal sample interface not reliant on dichroic elements and/or separation of the excitation and emission collection optics.

BRIEF SUMMARY OF THE INVENTION

[0016] Embodiments of the invention described herein are based on the inventive idea that, while requiring a first monochromator for selecting an excitation wavelength and a second monochromator for selecting a different emission wavelength, these two monochromators do not necessarily have to be independent of each other. According to embodiments of the present invention, it is possible to co-use several of the optical components of the excitation monochromator, namely the optically dispersive element and the exit aperture. They may concurrently constitute desirable parts of the emission monochromator. Following this concept, the exit aperture of the excitation monochromator also becomes the entrance aperture for the collected fluorescent light, which is then spectrally dispersed at the same optically dispersive element used to spectrally disperse the excitation radiation. For example, the same diffraction grating can be used to select the excitation wavelength as well as detect the emission wavelength. As the excitation and emission wavelengths are different, the emission radiation leaves the joint excitation/emission monochromator through a different aperture than the aperture through which the excitation radiation enters. Using a suitable optical design, as detailed below, this can be used to realize a compact dual-pass monochromator for excitation and emission.

[0017] As the dual pass monochromator' s excitation wavelength exit aperture and its emission radiation entrance aperture are identical, this device can be directly coupled to the sample using only one confocal beam path for excitation and emission.

[0018] Together, this allows for miniaturizing the overall system and reduces the number of optical components to a minimum while maintaining the performance and flexibility of a grating monochromator-based fluorescence assay reader.

[0019] In some embodiments, a dual pass monochromator uses a concave grating mirror as an optically dispersive element and is incorporated into a two double-monochromator layout, with the dual pass monochromator simultaneously acting as the second monochromator stage of the excitation double monochromator and as the first monochromator stage of the emission double monochromator.

[0020] The novel features and advantages of the invention, as well as the underlying principles of the invention itself, both as to its structure and operation, will best be understood from the accompanying drawings and the following examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 illustrates the basic optical layout of the dual-pass monochromator for excitation and emission radiation according to an embodiment of the present invention. In one embodiment, a concave grating mirror is used as an optically dispersive element.

[0022] Fig. 2a and Fig. 2b show other monochromator embodiments. Methods on how to adjust the settings of the dual-pass monochromator to different excitation and/or emission wavelengths are described below.

[0023] Fig. 3 shows the optical layout of another embodiment of the present invention. It is an integrated dual pass excitation/emission double/double monochromator in an exemplary all-grating arrangement with optical interfaces to an external sample and an external detector, respectively.

[0024] Fig. 4 shows an alternative embodiment to Fig. 3, where the first stage grating monochromator of the excitation double monochromator is replaced by a filter, such as a graded filter. Also shown is an alternative embodiment of the sampling optics that use short focal length lenses optimized for fluorescence light over a wide solid angle.

[0025] Fig. 5 illustrates an alternative compact geometrical layout based on the principle shown in Fig. 4, with desired stacking of the different beam sections on one another. In this case, three levels are connected by optical apertures. In this example layout, the detector is integrated directly into the monochromator.

[0026] The figures have been somewhat simplified for greater clarity. For example, only the beam paths for the prescribed excitation and emission wavelengths are shown, and some optical components, like plane mirrors used in the stacked arrangement (Fig. 5) to transfer the beams from one level to the next have been omitted. However, the behavior of undesired spectral components and the use of simple mirror optics to direct the beams in certain directions will be readily apparent to anyone with basic technical knowledge in optics.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Embodiments of the invention utilize the basic principles of a dispersive, or gratings-based, monochromators. As illustrated in Fig. 1, polychromatic excitation radiation 911 (e.g., light) having a range of wavelengths ∑_EX enters the monochromator through an entrance aperture Al in a housing 151. The radiation 911 is projected onto an optically dispersing element 11, which disperses the incident radiation 911 according to its wavelength. For a given setting of the monochromator, only radiation 912 with the chosen excitation wavelength X_EX leaves the monochromator through the exit aperture A2 in the housing 151, while the remaining undesired spectral components are discarded. The essentially monochromatic excitation radiation 912 can then be used e.g. to excite fluorescence in a sample 40.

[0028] Embodiments of the invention can advantageously can use the same monochromator to simultaneously spectrally analyze the fluorescence radiation 921 emitted by the sample 40. The polychromatic fluorescence radiation 921 emitted by the sample can include a range of wavelengths XE™ that are of equal or longer wavelength than the excitation wavelength X_EX. According to an embodiment of the present invention, the fluorescence radiation 921 can be directed into the monochromator through the same aperture A2 that previously acted as exit aperture for the monochromatic excitation radiation 912. The collected fluorescence radiation 921 is then projected, in a reverse direction to the excitation radiation 911/912, onto the optically dispersive element 11, and is spectrally dispersed according to its wavelengths. Only radiation 922 with the prescribed wavelength λεm_> corresponding to the emission wavelength to be measured, can leave the monochromator through a second exit aperture A3. In fluorescence measurements, the emission wavelength λ_Em is different from the excitation wavelength X_EX. Hence, as shown in Fig. 1 , the position of the second exit aperture A3 is spatially separated from the first entrance aperture Al. Using such a layout, it is possible to reliably separate the excitation and emission wavelengths for wavelength differences of typically approximately 20 nm or more.

[0029] The exemplary layout shown in Fig. 1 can effectively include two monochromators in one, i.e. an excitation monochromator comprising the entrance aperture Al, the optically dispersive element 11 and the exit aperture A2, and an emission monochromator comprising the entrance aperture A2, the optically dispersive element 11 and the exit aperture A3. This dual pass simultaneous use of the diffractive element 11 and the aperture A2 reduces the number of optical elements to a minimum number, and thus allows for realizing a highly compact excitation/emission monochromator. Furthermore, the interface between the excitation/emission monochromator and the sample can use confocal excitation/emission optics without dichroic or other wavelength selecting elements in them.

[0030] The disclosed principle is basically applicable with any suitable optically dispersive element 11, in particular prisms, gratings and prism-grating-combinations. For known practical reasons, in some embodiments, a suitable reflective diffraction grating is used as optically dispersive element 11. Consequently, this principle can be incorporated into most grating monochromator layouts previously described, e.g. the classical Czerny-Turner arrangement. Furthermore, most advantageous measures to improve the monochromator performance, like using suitably blazed gratings, can also be employed here.

[0031] In order to keep the number of optical elements as low as possible, the layouts shown in the accompanying figures can use a concave grating mirror as optically dispersive element 11. Concave grating mirrors with a VLS (varied line spacing) grating can also be used. Having both diffractive and focusing properties, no additional optical elements (e.g., collimation mirrors, etc.) are typically required when concave grating mirrors with VLS are used. [0032] A feature of embodiments of the invention is that a layout with fixed relative positions of the three apertures Al, A2 and A3 is applicable for only one specific excitation/emission wavelength combination. While the selection of either the excitation or the emission wavelength can easily be accomplished by angular displacement of the optically dispersive element 11 , the other wavelength is fixed by the thus determined incidence angle and the spatial position of the respective aperture.

[0033] One possibility to work around this restriction would be to make at least one of the separated apertures, i.e. the excitation entrance aperture Al or the emission exit aperture A3, positionable in the monochromator plane. While comparatively easy to realize for the aperture itself, the practical problem with this solution is that this movement would need to also involve the optics on the other side of the aperture, in this case the light source or the detector, and all optical components in between.

[0034] One solution is to use mirror optics to change the apparent position of an aperture Al or A3 relative to the optically dispersive element 11. The concept to achieve this is outlined in Figs. 2a and 2b for a layout affecting the beam path of the excitation radiation 911.

[0035] Fig. 2a shows the basic optical layout of a dual pass monochromator according to an embodiment of the present invention, which is set for simultaneously extracting a prescribed excitation wavelength X_EXI from the illumination radiation 911 and for extracting a prescribed emission wavelength λ_Emi from the collected fluorescence radiation 912. The difference with respect to Fig. 1 is the use of a tilted mirror 12 located between the entrance aperture Al and the optically dispersive element 11. In this layout, this plane mirror 11 serves also to achieve a better spatial separation of the first entrance aperture Al and the second exit aperture A3.

[0036] When adjusting the dual path monochromator to a different set of excitation and emissions wavelengths, as shown in Fig. 2b for an increased wavelength difference between excitation and emission wavelengths X_EX2 and λE_m2, the first step is to put the optically dispersive element in a proper position to select one of the two wavelengths. In the example shown, this is the emission wavelength X_E1112. To correctly select the other wavelength, in this example the excitation wavelength X_EX2, the incidence angle of the radiation has to be changed. Rather than changing the position of the corresponding entrance slit Al, this can be achieved by moving the intermediate mirror 12. By adjusting both the angular and lateral position of mirror 12 relative to the excitation beam path 911, the apparent position of the of the affected aperture, here the entrance aperture Al, can be changed relative to the optically dispersive element 11. Although not specifically illustrated, any suitable tilting and lateral movement mechanism may be used to move mirror 12.

[0037] This principle can be employed equally for either the excitation beam path 911 or the emission beam path 922. For special applications it may even be useful to employ the described principle of changing the apparent position of an aperture relative to an optically dispersive element by introducing a plane mirror that can be positioned both laterally (e.g., translatory movement) and angularly (e.g., rotational movement) in both beam paths 911 and 922.

[0038] It is now apparent to anyone knowledgeable in the field that the change of the mirror 12's position along the beam path of the focused beam, as shown in Figs. 2a and 2b, will also affect the position of the beam's focus. However, if appropriately selected optical elements are used, this effect proves negligible when the typical bandwidths of the excitation and emission wavelengths and aperture sizes are taken into account.

[0039] When building a fluorescence analyzer according to embodiments of the present invention, it may be desirable to further improve the stray and false light suppression and thus the achievable blocking ratio. One solution to this problem is to add a first single-stage monochromator for the excitation radiation 91 before the first entrance aperture Al and a second single-stage monochromator for the emission radiation 92 behind the second exit aperture A3, thus turning the dual pass monochromator into an integrated excitation/emission double/double monochromator.

[0040] Some embodiments of this concept are detailed in Figs. 3 and 4.

[0041] Fig. 3 shows the basic layout of an integrated excitation/emission double/double monochromator using the dual pass monochromator according to an embodiment of the invention as the core element, while using additional single grating monochromators for both additive monochromator stages.

[0042] In the embodiment in Fig. 3, the excitation radiation 91 is generated using a polychromatic light source 21 (e.g. a thermal source or a light emitting diode (LED)), and passes to a mirror optic 81. The focused light from mirror optic 81 passes through an aperture A4 into the first monochromator stage of the excitation double monochromator. The light of the chosen wavelength is then forwarded, using optical element 22, through aperture Al into the dual pass monochromator including mirror 12 and optically dispersive element 11. The radiation is spectrally separated again by the dual pass monochromator acting as the second stage of the excitation double monochromator. The radiation is then directed into the sample interface optics 31 and 41 in Fig. 3 (or 31 and 42 in Fig. 4) through the joint exit/entrance aperture A2.

[0043] The fluorescence radiation 92 emitted by the sample 40 upon excitation with the excitation radiation 91 is collected by the same sample interface optics 31 and 41 in Fig. 3 (or 31 and 42 in Fig. 4) and is transferred through aperture A2 into the dual pass monochromator, which is now acting as the first stage of the emission double monochromator. The relevant emission wavelength selected by the dual pass monochromator is forwarded through aperture A3 to the second monochromator stage, and from there to optically dispersive element 51 and through its exit aperture A5. The transmitted radiation is then directed to suitable detector interfacing optics 61, 71 and to the detector 70. A computational apparatus (not shown) including a processor, and a memory and an output and input devices operatively coupled to the processor, may be operatively coupled to the detector 70. The computational apparatus may analyze or output data generated by the detector 70.

[0044] When adjusting the prescribed excitation and emission wavelengths, the individual optically dispersive elements 11, 22 and 51 are brought into appropriate positions by angular movement, hi addition, the apparent position of aperture Al relative to the optically dispersive element 11 is adjusted by combined translatory and angular movement of the tilted mirror 12, as outlined above. As noted above, the mechanisms for moving the various optical elements (e.g., 11, 22, and 51) are not explicitly described herein, as they can be readily created by those of ordinary skill in the art.

[0045] An alternative embodiment of the invention is shown in Fig. 4. Some of the elements in FIG. 4 have been described above and the descriptions need not be repeated. In this layout, the first monochromator stage of the excitation double monochromator uses a non-dispersive element, i.e. some kind of filter. In some embodiments, a variable non- dispersive element, such as a variable filter (a graded filter 23), can be used, i.e. a tunable filter as shown in Fig. 4. The wavelength selection in this first monochromator stage is achieved be adjusting the pass wavelength of the filter, in the example shown, by moving the graded filter to a suitable position over the aperture Al leading into the dual pass monochromator acting as second stage of the excitation double pass monochromator.

[0046] Based on these two principles, other permutations of the disclosed dual pass monochromator with different coupled monochromator stages are also possible, hi other embodiments, the dual pass monochromator can also be used as part of a triple monochromator system. Such embodiments would be of interest for applications requiring a good spectral resolution and high blocking ratios. As outlined below, in addition to improving the spectral resolution, the disclosed dual pass monochromator could be used as a variable excitation/emission splitter in a confocal layout, thus replacing various optical elements, e.g., dichroic elements.

[0047] In yet other embodiments, all additional grating monochromator stages coupled to the dual pass monochromator can use concave grating mirrors 22, 51 additively coupled to the concave diffraction grating 11 used as an optically dispersive element in the dual pass monochromator. When using a set-up involving graded filters 23, some embodiments make use of graded filters with a filter gradient aligned to the dispersion of the optically dispersive element 11.

[0048] To keep stray and false light at lowest possible levels, all spectrometer stages can be contained in separated optical compartments 10, 15, 20, 30, 40, 50, 60 optically connected only by the defined apertures Al, A2, A3, A4, A5. The compartments may be defined by the outer and inner walls of a housing. The inner walls of these optical compartments can be covered with a suitable light absorbing material to minimize light scattering.

[0049] Fig. 5 shows the concept of a layout of the excitation/emission double/double monochromator based fluorescence analyzer in an embodiment with a minimized footprint, according to one embodiment of the present invention. Such embodiments can be used as an exchangeable or modular cartridge in a multi-purpose assay reader. The optical components can be arranged in several stacked levels. Fig. 5 illustrates this for a system according to Fig. 4, distributing the components on three superposed levels. Level I can include the radiation source 21 and the graded filter 23, can both be located in the light source compartment 20. Mirror optic 82 can also be included in Level I, and can receive radiation from the radiation source 21 and pass it to the graded filter 23. Level II, optically connectable to Level I via aperture Al, can include the dual pass monochromator in its compartment 10, and the sample interface optic 31 in compartment 30. The sample interface optic 31 and the optical element 41 can provide excitation radiation to and receive emission radiation from the sample 40. Aperture A2 optically couples compartments 10 and 30. Compartment 10 can include the previously described tilted mirror 12 and the optically dispersive element 11. The third superposed level, Level III, optically connectable to Level II via aperture A3, can include the second monochromator stage of the emission double monochromator in its optical compartment 50 and, in this example, the detector 70 can be located adjacent to aperture A5. Radiation from aperture A3 is received at concave grating mirror 51, and is then provided to mirror optic 83, before it exits aperture A5.

[0050] Building a compact instrument, on one or on several stacked levels, can make it desirable to add further mirror optics 81, 82, 83, e.g. for beam folding, beam shaping or vertical interfacing between different levels. These are readily apparent to someone knowledgeable in the field and do not change the scope of the invention.

[0051] In addition to enabling building a compact fluorescence analyzer, the second main advantage of various embodiments is that a confocal interface of the fluorescence analyzer to the sample without dichroic elements is possible.

[0052] With the exit aperture of the excitation light and the entry aperture of the emission light being the identical aperture A2, the same optics can be used to transfer the excitation light to and the fluorescence signals from the sample. Figs. 3 to 5 show a particular embodiment of such optics for use with micro-titer plates. The excitation light 91 coming from the exit aperture A2 is collimated using optics 31 and forwarded to the sample reader optics 41 or 42. These sample reader optics 41 or 42 determine the working distance to the sample 40 and the solid angle over which fluorescence light is collected. Fig. 4 shows yet another embodiment with sample reader optics 42 having a high numerical aperture, thus collecting fluorescence light over a wide solid angle. This layout is particularly advantageous for reading micro-titer plate wells through their bottoms, while for measuring through the top of the micro-titer plate well, a longer work distance, as shown in Figs. 3 and 5, may be more advantageous.

[0053] As illustrated in Fig. 3 and 4, a similar optical interface 61, 71 can also be used to connect the instrument to an external detector 70, preferably a photo-multiplier tube (PMT). Alternatively, it is equally possible to incorporate the detector 70 into the instrument, as illustrated exemplarily in Fig. 5.

[0054] It is apparent to one skilled in the art that various changes and modifications can be made to this disclosure, and equivalents employed, without departing from the spirit and scope of the invention. Elements shown with any embodiment are exemplary for the specific embodiment and can be used on other embodiments within this disclosure.

Claims

WHAT IS CLAIMED IS:

1. A dual pass monochromator comprising: a housing comprising an entrance aperture (Al), a first exit aperture (A2), and a second exit aperture (A3); a radiation source configured to provide radiation through the first entrance aperture (Al); and an optically dispersive element (11) configured to direct radiation of a first wavelength through the first exit aperture (A2) to a sample (40) and accept radiation of a second wavelength emitted by the sample (40) through the first exit aperture (A2) and direct the radiation emitted by the sample (40) through the second exit aperture (A3), wherein the second exit aperture (A3) is spatially separated from the entrance aperture (Al).

2. The dual pass monochromator according to Claim 1 wherein the optically dispersive element comprises a diffractive element.

3. The dual pass monochromator according to Claim 2 wherein the diffractive element is a concave grating mirror.

4. The dual pass monochromator according to Claim 1 further comprising means for effecting an angular displacement of the optically dispersive element to select one or more prescribed wavelengths.

5. The dual pass monochromator according to Claim 4 further comprising means for moving the spatial position of the entrance aperture (Al) or the second exit aperture (A3) to select prescribed wavelengths.

6. The dual pass monochromator according to claim 4 further comprising a plane mirror capable of both translatory and rotational movement and located between the entrance aperture (Al) and the optically dispersive element (11) as means to change the apparent position of the entrance aperture relative to the optically dispersive element.

7. The dual pass monochromator according to Claim 4 further comprising a plane mirror capable of both translatory and rotational movement and located between the optically dispersive element (11) and the second exit aperture (A3) as means to change the apparent position of the optically dispersive element (11) relative to the second exit aperture (A3).

8. An excitation/emission double/double monochromator comprising the dual pass monochromator of Claim 1 wherein the dual pass monochromator is used as both a second stage of an excitation double monochromator and a first stage of an emission double monochromator.

9. The excitation/emission double/double monochromator according to Claim 8 further comprising an optically dispersive element in a first stage of the excitation double monochromator or a second stage of the emission double monochromator.

10 The excitation/emission double/double monochromator according to Claim 9 wherein the optically dispersive element comprises a reflective diffraction grating.

11 . The excitation/emission double/double monochromator according to Claim 10 wherein the reflective diffraction grating comprises a concave grating mirror.

12. The excitation/emission double/double monochromator according to Claim 9 wherein diffraction gratings are aligned to additively couple to the optically dispersive element.

13. The excitation/emission double/double monochromator according to Claim 8, further comprising non-dispersive wavelength selectors in a first stage of the excitation double monochromator or a second stage of the emission double monochromator coupled to the dual pass monochromator, wherein the non-dispersive wavelength selectors comprise bandpass filters, tunable filters or graded filters.

14. The excitation/emission double/double monochromator according to Claim 8, further comprising a variable non-dispersive element in a first stage of the excitation double monochromator, and a second optically dispersive element additively coupled to the optically dispersive element of the dual pass monochromator in a second stage of the emission double monochromator, wherein the variable non-dispersive element comprises a graded filter and the second optically dispersive element comprises a concave grating mirror.

15. A fluorescence reader comprising the excitation/emission double/double monochromator according to Claim 8.

16. The fluorescence reader according to Claim 15 for reading the fluorescence in wells of a micro-titer plate, wherein the same optics are used for confocally illuminating the sample with the excitation radiation, collecting fluorescence radiation, and transferring the collected fluorescence radiation back to the monochromator.

17. The fluorescence reader according to Claim 16 for reading the fluorescence emitted by samples in wells in a micro-titer plate through the bottom of the wells, wherein illumination/collection optics with short focal length and high numerical aperture are used at the sample interface to collect a possibly high amount of the fluorescence emitted by the sample.

18. The fluorescence reader according to Claim 15 having a compact footprint, for use as a modular fluorescence analyzer in a multi-purpose assay reader, wherein the fluorescence reader comprises different optical compartments, as defined by separated optical chambers optically coupled only through apertures, on at least two stacked layers.