Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
  • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
  • G01J3/28—Investigating the spectrum
  • G01J3/2803—Investigating the spectrum using photoelectric array detector
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
  • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
  • G01J3/28—Investigating the spectrum
  • G01J3/30—Measuring the intensity of spectral lines directly on the spectrum itself
  • G01J3/36—Investigating two or more bands of a spectrum by separate detectors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

• optical signaling events that have different optical characteristics which may then be identified and potentially quantified separately from each other's optical signals.
• analytical assays include medical diagnostic tests, food and other industrial process analyses, and basic tools of biological research and development. While a wide variety of optical and chemical approaches have been applied toward analysis of these signals, such systems often include a level of complexity and/or cost that detracts from the overall utility of the approach, particularly for operations that require high levels of sensitivity.
• the present invention addresses these shortcomings of other systems and methods.
• the present invention generally provides methods and systems for detecting and monitoring a plurality of different optical signals from a single, preferably confined source of such signals.
• such systems and methods are applied to the detection of luminescent or fluorescent signals from fluid borne materials and particularly reactants and/or products of chemical, biochemical or biological reactions of interest.
• the present invention provides methods of detecting optical signals, where such methods comprise providing a source of at least first and second optical signals wherein the first optical signal comprises an optical characteristic different from an optical characteristic of the at least second optical signal.
• the optical characteristic is a wavelength of the optical signals.
• the optical signals are directed to different locations on a detector, e.g., by passing the signals through an optical train that transmits the first and second optical signals in divergent paths, and then received at different locations on one optical detector.
• the method of detecting optical signals comprises providing a source of a plurality of different optical signals, wherein each different optical signal comprises a wavelength different from each other optical signal, and spatially separating the plurality of different optical signals and directing them to discrete locations on one optical detector.
• a method for detecting optical signals comprises providing a confined source of at least first and second optical signals wherein the first optical signal comprises a different optical characteristic, i.e., wavelength, from that of the at least second optical signal. The signals are then spatially separated and directed to first and second different locations on a first optical detector.
• the present invention also provides for systems useful in carrying out the foregoing methods.
• the invention provides analytical systems, comprising a confined reaction region for containing a reaction mixture that produces at least first and second optical signals wherein the first optical signal comprises an optical characteristic different from that of the at least second optical signal.
• Such systems also comprise an optical train in optical communication with the confined reaction region, for receiving the first and second optical signals and spatially separating the first and second optical signals and directing them to different locations on an optical detector.
• Related systems of the invention comprise a confined reaction region for containing a reaction mixture that produces at least first and second optical signals wherein the first optical signal comprises a wavelength different from a wavelength of the at least second optical signal, an optical train in optical communication with the confined reaction region, for receiving the first and second optical signals and spatially separating the first and second optical signals and directing them to different locations on an optical detector.
• the optical train comprises a replaceable modular optical component that spatially separates the first and second optical signals passing therethrough.
• Figure IA provides a simplified schematic illustration of the methods and system of the invention.
• Figure IB schematically illustrates separation of optical signals from the system of Figure IA.
• Figure 2 provides a schematic illustration of the operation of the systems and methods of the invention in monitoring multiple different optical signals over time.
• Figure 3 schematically illustrates one exemplary system according to the present invention in greater detail.
• Figure 4 schematically illustrates an alternate system configuration for monitoring multiple optical signals that differ in their relative polarization, as opposed to other characteristics of light, e.g., wavelength.
• Figure 5A shows different optical signals incident upon different locations of a single CCD camera chip, which were derived from a single, combined source, and subjected to the methods of the invention.
• Figure 5B shows the relative distance of separation between separated signals.
• the present invention is generally directed to devices, systems and methods for the facile, efficient and cost effective analysis and/or management of collections of optical signals and the data derived from those signals.
• devices, systems and methods for the facile, efficient and cost effective analysis and/or management of collections of optical signals and the data derived from those signals.
• reactions of interest e.g., chemical and biochemical reactions such as nucleic acid synthesis, and the characterization of the steps involved in those reactions.
• the present invention is directed to methods, systems and devices for measuring two or more different optical signals from a source of optical signals, by separating the optical signals from each other and directing them to different detection functionalities, or different locations, on a single optical detector.
• the present invention By separately detecting the different optical signals one can recognize the occurrence of the causal events for each signal.
• one can reduce the complexity and cost of systems and their associated control and analysis processes, while concurrently increasing their efficiency and/or sensitivity.
• a wavelength of an optical signal includes a wavelength range for that signal.
• optical signals e.g., emitted fluorescence, luminescence, or the like, will span a portion of the optical spectrum which portion may span a range of from 1 nm to 30 nm up to 100 nm or more within the overall spectrum.
• optical signals of different wavelengths denote signals whose wavelength range is distinguishable from the other.
• the analytical methods and systems of the invention are applied in nucleic acid analyses and particularly nucleic acid sequence analyses.
• the methods and systems of the invention have reduced complexity, and as a result, higher sensitivity, they are particularly useful in applications where the optical signals to be detected are relatively weak, e.g., low light levels, few signal events, etc.
• the systems employed in the invention minimize the number of optical manipulations that signals are put through, the overall efficiency losses of the system that are summed from each such manipulation are likewise reduced. For example, where optical signals are passed through multiple beam splitting, refocusing, filtering, etc. operations, losses associated with each stage can dramatically reduce the sensitivity of the overall assay.
• losses associated with examining only a separate portion of the optical spectrum of the overall signal can further reduce the amount of signal that could otherwise be used in the detection operation.
• the entire spectrum of the overall signal is subjected to detection, and selection of each different signal component is a matter of selecting the location on a single detector, e.g., which pixels in a detector array, should be applied toward assessing a given signal, rather than cutting off a portion of signal before it is ever detected through, e.g., optical cut-off filtering.
• the invention is directed to methods of detecting optical signals, from a source of a plurality of different optical signals, by separating the different optical signals from each other and directing at least a portion of them to discrete locations on one optical detector or detector array.
• detector includes or is capable of being configured to provide signal information for signals incident thereon, that correlate not only the signal intensity and time, but also the position or location upon the array at which such signal is incident.
• Simple examples of such detectors include array type detectors as are generally known in the optics art, and certain examples of which are described in greater detail herein.
• position information of an incident signal is provided by the location of each individual detector (typically although not necessarily of a plurality of individual detectors), rather than a location within one single detector or detector array.
• the source of optical signals comprises a confined source.
• the confined sources of the inventions are typically characterized in that one or more components of the source that produce the particular optical signals are confined in space, and are not flowing into and or out of the confined source during the detection.
• Such confined sources are in contrast to systems where signal producing components, reactants, or the like are actively flowing past a point of detection in a conduit.
• components of the signal producing mechanism employed in the invention may be diffusing into and out of the confined space, while still falling within the parameters set forth herein. In many cases, however, one or more components that contribute to the signaling mechanism will be immobilized within the confined space.
• the confined nature of the sources is of particular value where the optical signals result from reactive chemical species and particularly fluid borne reactive chemical species, e.g., aqueous and/or organic fluids.
• the confined nature of the source would not permit the movement of such fluids into or out of the confinement during detection.
• fluid confinements include, e.g., conventional multiwell analysis plates, e.g., 96, 384 or 1536 well plates.
• confinements for such fluid reactants include nanoscale wells or apertures, i.e., zero mode waveguide structures as described in Published U.S. Patent Application No.
• This fractional observed volume represents a further confinement of the signal source.
• confined volumes in single molecule interactions, such as DNA sequence identification through the stepwise reaction of labeled nucleotide analogs with a nucleic acid polymerase in template dependent nucleic acid synthesis, molecular interaction monitoring, i.e., DNA hybridization, immunoassays, enzymatic reactions, and the like.
• confinement may additionally or alternatively comprise chemical immobilization of chemical species that produce one or more of the optical signals, i.e., either in place of or in addition to any structural confinement.
• chemical confinement include covalent, van der waals or other associative interactions between chemical species and substrate surfaces, use of chemical interactions to create structural confinements, e.g., substrates having hydrophilic regions surrounded by hydrophobic barriers to confine fluid and chemical species, and the like.
• confinement denotes chemical immobilization of reactants in a given location
• immobilization techniques including, e.g., covalent linkage of reactants onto surfaces of supports or substrates, including for example silane or epoxide linkages.
• other associative linkages may be employed using, e.g., complementary binding pairs to couple reactants to substrates or supports.
• linkages include, e.g., antibody/antigen linkages, biotin/avidin linkages, and the like.
• a variety of techniques are available for providing such 'structures' on substrates.
• hydrophobic barriers may be created by providing alkylsilane groups on otherwise hydrophilic silica surfaces. Such materials are readily patterned onto substrate surfaces using conventional photolithographic techniques, screen printing, ink-jet printing or the like, to define hydrophilic confines surrounded by hydrophobic barrier regions.
• the optical signals emanating from the source derive from reactive chemical species, where the reaction of such species either produces, extinguishes, increases, decreases, or otherwise alters the characteristic of the optical signals.
• reactive species include chromogenic or chromophoric reactants, e.g., that produce a shift in the transmissivity of the material to light of one or more wavelengths, i.e., changing color upon reaction.
• Reactant species that emit light either with the use of an activating light source (fluorescent or fluorogenic) or without such an excitation source (luminescent) are preferred for use in the methods of the invention.
• such reactive species are most preferably contained in fluid solutions and are provided as reaction mixtures where the different optical signals result from the substrates, the products, or combinations of the two.
• the different optical signals to be detected are comprised of light of differing wavelengths, e.g., emitted by different fluorophores where such emissions have different wavelength spectra, or transmitted by different chromophores where such transmissions are at different wavelength spectra.
• the two or more different optical signals are spatially separated, e.g., through the use of a beam splitter in combination with one or more dichroic filters, or through the use of a prism or optical grating, and the different signals are directed to different locations on an optical detector or detector array.
• the different optical signals may differ in other characteristics, such as their relative polarity, their modulation phase or frequency, or the like, provided that they may be spatially separated and directed to different regions on a detector or detector array, e.g., through the use of polarizing or demodulation filters.
• biochemical assays based upon such differing characteristics are described in, e.g., U.S. Patent No. 6,699,655, which discloses monitoring reaction progress by detecting of the relative polarity of fluorescent reactants and products (typically in combination with a polarization affecting agent) when excited with polarized light.
• the methods of spatial separation and/or direction of different optical signals to different locations on an optical detector or detector array is generally dependent upon the characteristic(s) of the different optical signals that is/are to be the basis of differential detection.
• separation and direction can be accomplished through the use of optical filters and/or prisms that selectively transmit or redirect light of differing wavelengths in different manners and/or to different degrees.
• a collected signal that comprises two different wavelengths of light emanating from a confined source may be split into two beams, e.g., through the use of a dichroic filter to remove the other signal component, then passed through a barrier filter, thereby allowing only a portion of the overall signal to be directed to the optical detector or detector array.
• a simpler optical train is employed to separate optical signals and direct them to different locations on a detector or detector array, or in some cases, to multiple different detectors or detector arrays.
• a wedge prism or optical grating may be employed to achieve this result.
• the use of such prisms or diffraction gratings provides simplicity to the optical train of the overall system and results in a more transmissive light path as compared to more complex optical systems.
• cut-off filters e.g., dichroics
• the entire spectrum of signal, or at least a more selectively filtered portion of the signal, less, e.g., the reflective losses of the prism may be directed to the detector or detector array.
• the component of the optical train that spatially separates the optical signals may comprise a modular, and easily replaceable component, such as a prism, multiple prisms, and/or optical grating(s), that can be inserted into and ejected from an appropriate receiver slot on an instrument.
• a given instrument may be supplied with ort suppliable with a library of such modular components, where each of the components provides different optical dispersion profiles for different optical signals or collections of optical signals, allowing facile reconfiguration of the separation component by the end user and maximal usefulness and flexibility to the user.
• n optical signals where n>l
• detection of n optical signals is typically accomplished through the use of at most, n-1 discrete detectors.
• as many as 2, 3, 4, 5, 6 or more different optical signals are directed to different locations on 1, or in cases of 3 or more signals, 2 or more discrete optical detectors or detector arrays.
• detectors are not single point detectors, e.g., simple photodiodes, but instead have a detection area that generates a signal that is indicative of the incidence of an optical signal on the detector, as well as an indication of the location on the detector where such signal was incident.
• detectors include imaging detectors, such as charge coupled devices (CCDs), where each pixel element on the CCD constitutes a single point detector, but the overall device constitutes an array of detectors, where the detector signal indicates the pixel at which the signal was incident and the intensity of that signal at that pixel.
• CCDs charge coupled devices
• larger diode array detectors may be used that include larger numbers of photodiodes spatially arranged and interfaced to provide both signal intensity and signal location information within the array.
• simple point detectors may be used in conjunction with such detector arrays in accordance with the invention, e.g., where single signals are directed to a single detector, and different signals are directed to different, or discrete detectors, rather than to regions on the same detector.
• each different signal is optionally directed to a different detector element, e.g., a point detector.
• a detector element e.g., a point detector.
• the incidence of an optical signal at a particular location on the detector or detector array indicates that one of the two optical signals is being emitted or transmitted from the confined source. If two or more locations on the detector or elements on the detector array indicate the incidence of an optical signal, it is indicative that two or more different optical signals are being emitted.
• FIG. 1A A simplified schematic of the methods of the invention is illustrated in Figure IA.
• At least two different optical signals 102 and 104 emanate from a confined source 106 of such signals.
• confined sources may preferably be defined locations that comprise fluid borne chemical reactants, such as reaction wells or regions, zero mode waveguides, etc.
• the different optical signals are then spatially separated (as shown by the divergent paths of solid arrows 102 and dashed arrows 104) by passing those signals through an appropriate optical component, e.g., prism 108, an optical grating or the like. Once separated, the signals are focused through lens 110, e.g., an imaging lens, causing them to impinge on detector array 112 at two different locations 114 and 116 on that detector array 112.
• lens 110 e.g., an imaging lens
• Figure IB The separation of signals is illustrated schematically in Figure IB.
• the combined optical signals enter prism 108 as a signal as represented by spot 150.
• the signals Once the signals have passed through the spatial separation component of the optical train, e.g., prism 108, and are focused onto the detector, they are spatially separated into their respective different optical signal components, as represented by spots 152 and 154.
• Figure 2 schematically illustrates the detection operations over a period of time, where the signals are concurrent or not.
• the system 100 is further connected to a recording/readout system, schematically illustrated as plot 202.
• optical signals emanate from the confined source 106, either at different times (as shown at times 204 and 206) or concurrently (at time 208).
• the optical signals are detected on different locations of the detector 112, where each location is separately connected to the recording system (e.g., at connections 210 and 212).
• optical signals from a single confined source are separately detected and recorded, and can be attributed to a given point in time.
• One exemplary use of the methods of the present invention is in the performance of nucleic acid sequence analysis processes, and particularly single molecule based processes that analyze nucleic acid sequences by monitoring the template dependent synthesis of complementary nucleic acid sequences through the detection of differently labeled nucleotide analogs that are incorporated into the growing synthesized strand. See, e.g., U.S. Patent Application Nos. 2003/004478 IAl, which is incorporated herein by reference in its entirety for all purposes.
• a DNA polymerase enzyme is associated or complexed with a template nucleic acid sequence, which is immobilized on the surface of a substrate, attached through either the template or the polymerase.
• the complex is exposed to appropriate polymerization reaction conditions, including differently labeled nucleoside polyphosphates, e.g., nucleoside triphosphates (NTPs), nucleoside tetraphosphates, nucleoside pentaphosphates, etc., or analogs of any of these, or other nucleoside or nucleotide molecules, that are incorporated by polymerase enzymes (all of which are referred to herein as NTPs, for convenience), where each different NTP (e.g., A, T, G, or C) is labeled with fluorescent label having a different emission wavelength profile.
• NTPs nucleoside triphosphates
• NTPs nucleoside tetraphosphates
• nucleoside pentaphosphates etc.
• each incorporation signal is directed to a different location on an optical detector array, and identified based upon that location upon the detector array.
• the sequence of the immobilized sequence By identifying the probes that hybridize, e.g., remain localized, within the confined area of the immobilized nucleic acid, one can identify the sequence of the immobilized sequence. Likewise, where the immobilized sequence is known, one can identify the sequence of the probe sequences that hybridize to it.
• assays that detect differences in fluorescent polarization capabilities of substrate and product may be monitored using the methods and systems of the invention.
• U.S. Patent No. 6,699,655 which is incorporated herein by reference in its entirety for all purposes, describes homogeneous assay systems that are capable of monitoring reactions in which reactants and products have substantially different charges.
• Such assays include kinase or phosphatase assays where phosphorylated or dephosphorylated products have substantially different charges as compared to their substrates, as a result of addition or removal of a phosphate group, nucleic acid hybridization assays, protease assays, and the like.
• a large, charged molecule or other structure associates differentially with a substrate or product, based upon the charge differential, and thus changes the rotational diffusion of the substrate or product, consequently changing the relative polarization of fluorescence emitted from an attached fluorescent label in response to polarized excitation radiation.
• the two different signals are first spatially separated, and then directed to different locations on the same detector.
• An example of a system for use in performing applications that distinguish among different polarized optical signals is shown in Figure 4.
• FIG. 3 schematically illustrates one exemplary system for carrying out the methods of the present invention.
• the overall system 300 includes a source of at least two different optical signals 302.
• source 302 comprises a substrate that includes at least one, and preferably an array of zero mode waveguides 304 fabricated thereon.
• An optical train 306 is also provided that is in optical communication with the source 302, including waveguides 304.
• optical train 306 includes a source of excitation radiation, e.g., a laser 308, laser diode, LED, or the like, for use with fluorescent or fluorogenic optical signaling components within the source 302.
• a dichroic mirror 310 that reflects excitation radiation to direct it toward the source 302, e.g., including waveguide 304, but that will pass emitted fluorescence.
• An objective lens or other focusing lens 312 is also typically provided to focus and further direct excitation radiation to and optical signals, e.g., fluorescence, from source 302.
• the signal is passed through a barrier or notch filter 314 to further reduce any excitation radiation not reflected by dichroic 310, and then through a prism 316 or optical grating is provided to spatially separate excitation radiation by, e.g., wavelength, and direct it through lens 312, and onto an optical detector, e.g., CCD 320.
• Useful prisms and/or optical gratings are generally commercially available from a variety of commercial optics suppliers, including, e.g., Thorlabs, Inc. (New Jersey), Newport Corp (Irvine, California), CVI Corporation (Alberquerque, New Mexico), and the like.
• the signals detected upon CCD 320 are recorded by processor 322 which may perform one or more data manipulations on such recorded signal data (e.g., to assign a reaction parameter, etc.) and then provided in a user friendly readout format, e.g., on display 324.
• the spatial separation of different signals resulting from the dispersion profile of a given prism may not achieve a desired spatial separation.
• the dispersion profiles of given prism may not be linear, e.g., the resulting transmitted signals are not equally spatially separated.
• tuning of the system may be accomplished by rotating the prism or other dispersive optical element, e.g., around the optical axis of the optical system and also perpendicular to the direction of color separation, to adjust the degree of dispersion.
• the source of different optical signals 302 includes a reaction mixture that generates products, or consumes substrates that produce at least two different optical signals, e.g., substrates, intermediates and/or products that bear fluorescent labels that emit light at differing wavelengths.
• Light source e.g., laser 308, directs excitation radiation, e.g., light at an appropriate excitation wavelength for the fluorescent labels present in the source 302, toward dichroic 310.
• the excitation radiation is reflected by dichroic 310, through objective 312, to impinge upon the source 302, thus exciting the fluorescent labels contained therein.
• the emitted fluorescence is again collected by objective 312 and directed through dichroic 310, which is selected to reflect light of the wavelength of the excitation radiation, but pass light of the wavelength(s) of the emitted fluorescence. As a result, any reflected excitation radiation is filtered away from the fluorescence.
• the fluorescent signal(s) are then directed through a prism 316 or optical grating that spatially separates the differing signals by wavelength, and then refocused using a lens 318, e.g., an imaging lens, and directs them to different locations on an optical detector array, e.g., CCD 320, photon counting avalanche photodiode array, photomultiplier tube (PMT) array or the like.
• CCDs are generally preferred for their compact nature, high resolution and cost, and may generally be employed as the detector.
• Various types of CCDs may be employed to suit the needs of a given analysis, including, for example, standard CCDs, electron multiplier CCDs (EMCCD), and/or Intensified CCD (ICCD).
• FIG. 4 is a schematic illustration of a system that directs optical signals that differ from each other in the relative polarity of the emitted fluorescence.
• Such detection may be employed in monitoring reactions that yield substantial size changes on products or reactants, and consequently changes in the reactant or product's ability to emit depolarized fluorescence (See, e.g., U.S. Patent No. 6,699,655).
• U.S. Patent No. 6,699,655 See, e.g., U.S. Patent No. 6,699,655
• the system, 400 again includes an activation light source 402 that is directed through a dichroic filter 406 and objective 408 toward a confined reaction vessel or region 410.
• Light source 402 may comprise a polarized light source or be directed through a polarizing filter 404 to provide polarized excitation radiation to the reaction vessel 410.
• Emitted fluorescence is then collected by objective lens 408 and directed through beam splitter 412, where it is split into two similar beams. Each beam is then separately passed through one of two oppositely polarized filters 414 and 416, such that only fluorescence in one of the two orthogonal planes is passed through lens 418 to each of the regions 422 and 424 on detector array 420.
• each signal on the detector array is an indication of which plane of fluorescence is being detected.
• the intensity of the signals are then compared to determine the relative depolarization of fluorescence from the reaction mixture (See, again, U.S. Patent No. 6,699,655). IV. Examples
• a system was set up that was substantially similar to the system shown in Figure 3. As shown, the system included a substrate having a series of zero-mode waveguides fabricated thereon. The substrate was positioned proximal to and within optical communication of objective lens, and a white light source was positioned above the zero mode waveguide substrate and directed through a narrow band filter, at the waveguide substrate. An objective lens was used to focus optical signals from the waveguides through wedge prism. Once separated by wedge prism, the different optical signals were then passed through the imaging lens onto a 512 X 512 pixel EMCCD camera chip.
• FIG. 5A illustrates the images derived from four different regions of the CCD, corresponding to light from the eight different zero mode waveguides and four different wavelengths, 405 nm (A), 488 nm (B), 568 nm (C) and 647 nm (D).
• Figure 5B is a plot of the relative location, in distance from a position of an unseparated signal, in microns, showing the relative separation distance between the separated signals.
• a comparison experiment was also performed to demonstrate the increased efficiency of the prism based separation as compared to a filter based wavelength separation.
• a mixture of two different fluorescent dyes Alexa488 and Alexa568, available from Molecular Probes, Eugene, OR
• peak emission wavelengths 488nm and 568 nm, respectively
• Emissions from the mixture were passed through an objective and subjected to either filter based wavelength separation (using two Semrock triple notch filters, or wedge prism based separation, prior to focusing the separated signals onto a CCD chip.
• filter based wavelength separation using two Semrock triple notch filters, or wedge prism based separation, prior to focusing the separated signals onto a CCD chip.
• the table, below provides fluorescence intensities of each signal in each different optical train, as measured using an EMCCD.
• the prism based separation yields substantially higher efficiency detection of the separated signal as compared to the filter based system.

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