Patent Description:
Optical systems for biological and biochemical reactions have been used to monitor, measure, and/or analyze such reactions in real time. Such systems are commonly used in sequencing, genotyping, polymerase chain reaction (PCR), and other biochemical reactions to monitor the progress and provide quantitative data. For example, an optical excitation beam may be used in real-time PCR (qPCR) reactions to illuminate hybridization probes or molecular beacons to provide fluorescent signals indicative of the amount of a target gene or other nucleotide sequence. Increasing demands to provide greater numbers of reactions per test or experiment have resulted in instruments that are able to conduct ever higher numbers of reactions simultaneously.

The increase in the number sample sites in a test or experiment has led to microtiter plates and other sample formats that provide ever smaller sample volumes. In addition, techniques such as digital PCR (dPCR) have increased the demand for smaller sample volumes that contain either zero or one target nucleotide sequence in all or the majority of a large number of test samples. The combination of small feature size (e.g., an individual sample site or volume) and large field of view to accommodate a large number of test samples has created a need for optical systems that provide high optical performance with relatively small sample signals.

The reduction in sample volumes has also lead to a desire to incorporate light sources that provide a large amount output power or intensity. In recent years, advance in LED (Light Emitting Diode) technology resulted in availability of LED sources with significantly larger outputs. In addition, high power LED sources are now available with a broad spectrum, for example, white light LEDs that provide significant output power across the visible spectrum. Broad spectrum or white light LEDs are also attractive in biological applications such as PCR, since they allow for a broad range of dyes or markers to be used in a single sample or instrument. However, high power LEDs can have large power and spectral variations from the nominal specification. Thus, various LEDs having the same part number or output specification may result in unacceptably large instrument to instrument variation, particularly would couple with other system tolerance variation (e.g., variations in filter and beamsplitter optical characteristics). Thus, there exists a need for better control and calibration systems, devices, and methods when attempting to incorporate high power, broad spectrum LED into biological instruments.

<CIT> describes an optical instrument which illuminates two or more spaced-apart regions, and that comprises a light source and a collimating lens to form bundles of collimated excitation beams.

<CIT> describes a method and an apparatus of analyzing samples contained in a microplate. The instrument is capable of measuring fluorescence, luminescence, and/or absorption within multiple locations within a sample well and is tunable over the excitation and/or detection wavelengths.

<CIT> describes a system that includes a light-emitting diode, a temperature sensor in thermal contact with the light-emitting diode and capable of measuring an operating temperature and generating an operating temperature signal.

Embodiments of the present invention may be better understood from the following detailed description when read in conjunction with the accompanying drawings. Such embodiments, which are for illustrative purposes only, depict novel and non-obvious aspects of the invention. The drawings include the following figures:.

As used herein, the term "light" means electromagnetic radiation within the visible waveband, for example, electromagnetic radiation with a wavelength in a vacuum that is within a range from <NUM> nanometers to <NUM> nanometers. As used herein, the term "infrared" means electromagnetic radiation having a wavelength within a range of <NUM> micrometer to <NUM> micrometers.

As used herein, the term "optical power" means the ability of a lens or optic to converge or diverge light to provide a focus (real or virtual) when disposed within air. As used herein the term "focal length" means the reciprocal of the optical power. As used herein, the term "diffractive power" or "diffractive optical power" means the power of a lens or optic, or portion thereof, attributable to diffraction of incident light into one or more diffraction orders. Except where noted otherwise, the optical power of a lens, optic, or optical element is from a reference plane associated with the lens or optic (e.g., a principal plane of an optic).

As used here, the term "about zero" or "approximately zero" means within <NUM> of the unit of measure being referred to, unless otherwise noted. For example, "about zero meters" means less than or equal to <NUM> meters, if the dimension may only reasonably have a positive value, or within a range of -<NUM> meters to +<NUM> meters, if the dimension may have either a positive or negative value.

When used in reference to an optical power in units of Diopters, the terms "about" or "approximately", as used herein, means within <NUM> Diopter. As used herein, the phrase "about zero Diopter" or "approximately zero Diopter" means within a range of -<NUM> Diopter to +<NUM> Diopters.

Referring to <FIG>, a system or instrument <NUM> for biological analysis comprises an optical system <NUM>. In certain embodiments, system or instrument <NUM> additionally comprises a sample block or processing system <NUM> and/or a computer system, electronic processor, controller <NUM> configured to control, monitor, and/or receive data from optical system <NUM> and/or sample processing system <NUM>. Without limiting the scope of the present invention, system or instrument <NUM> may be a sequencing instrument, a polymerase chain reaction (PCR) instrument (e.g., a real-time PCR (qPCR) instrument and/or digital PCR (dPCR) instrument), an instrument for providing genotyping information, or the like.

The optical system <NUM> comprises an illumination or excitation source <NUM> providing one or more excitation beams <NUM> and an optical sensor or detector <NUM> configured to receive one or more emission beams <NUM> from one or more biological samples <NUM>. Excitation source <NUM> comprises and operates in conjunction with, an excitation source temperature controller <NUM>, used to maintain the temperature of excitation source <NUM> above or below a predetermined temperature. The system <NUM> also comprises an excitation optical system <NUM> and an emission optical system <NUM>. Excitation optical system <NUM> is disposed along an excitation optical path <NUM> and is configured to direct the electromagnetic radiation or light from excitation source <NUM> to sample holder containing one or more biological samples. Emission optical system <NUM> is disposed along an emission optical path <NUM> and is configured to direct electromagnetic emissions from biological samples <NUM> to optical sensor <NUM>, for example, one or more fluorescence signals produced at one or more wavelengths in response to the one or more excitation beams <NUM>. Optical system <NUM> may further comprise an emission filter assembly <NUM> comprising a plurality of filters, filter components, elements, or modules <NUM> configured to interchangeably locate or move one or more of filter modules <NUM> into emission optical path <NUM>. Optical system <NUM> may additionally comprise an excitation filter assembly <NUM> comprising a plurality of filters, filter components, elements, or modules <NUM>, wherein excitation filter assembly <NUM> is configured to interchangeably locate or move one or more of filter modules <NUM> into excitation optical path <NUM>. Optical system <NUM> may further comprise a first optical element <NUM> configured to direct light to optical sensor <NUM>, a second optical element <NUM> configured to direct excitation light to, and/or emission light from, the biological samples, a beamsplitter <NUM>, and/or one or more optical windows <NUM>.

In certain embodiments, sample processing system <NUM> comprises a carrier or support frame <NUM> configured to receive a sample holder <NUM>. Sample holder <NUM> comprises a plurality or array of cells <NUM> for containing a corresponding plurality or array of biological samples <NUM> that may be processed by sample processing system <NUM> and/or optical system <NUM>. Cells <NUM> may be in the forms of sample wells, cavities, through-holes, or any other chamber type suitable containing and/or isolating the plurality of biological samples <NUM>. For example, sample cells <NUM> may be in the form of sample beads in a flow cell or discrete samples deposited on top of a substrate surface such as a glass or silicon substrate surface.

With additional reference to <FIG>, sample holder <NUM> comprises <NUM> sample cells <NUM> that are in the form of <NUM> sample wells <NUM> configured to provide <NUM> isolated or distinct biological samples <NUM>. Alternatively, sample holder <NUM> may comprise less than <NUM> well and samples, for example, <NUM> wells and samples, or may contain more than <NUM> wells, for example, <NUM> or more wells and samples. In certain embodiments, carrier <NUM> is configured to receive more than one sample holder <NUM> for simultaneous processing by sample processing system <NUM> and/or optical system <NUM>.

Sample processing system <NUM> may further comprise a block or assembly <NUM> for receiving sample holder <NUM> and a sample thermal or temperature controller <NUM> for controlling and/or cycling the temperature of biological samples <NUM>. In certain embodiments, sample holder <NUM> includes all or a portion thermal controller <NUM>. Sample processor system <NUM> may further comprise a thermally controlled or heated lid <NUM> disposed about sample holder <NUM>. Thermally controlled lid <NUM> may be configured to aid in controlling a thermal and/or humidity environment of biological samples <NUM> or sample holder <NUM>, for example, to aid in preventing condensation from forming on samples <NUM> or optical elements of sample holder <NUM>. In certain embodiments, system <NUM> includes a set of different types or configurations of block <NUM> and/or different types or configurations of thermally controlled lid <NUM>, where each member of the set is configured for use with a different type or number of sample holders <NUM> or carriers <NUM>. Sample temperature controller <NUM> may comprise all or a portion of heated lid <NUM> and/or hardware used to control the temperature of, or heat flow into, heated lid <NUM>.

Referring to <FIG>, system <NUM> may be additionally or alternatively configured to receive and process a sample holder <NUM> comprising a substrate <NUM> including a plurality of through-holes <NUM>. In such embodiments, through-holes <NUM> are configured to maintain isolated or distinct biological samples <NUM> by capillary forces, for example, by forming through-holes to have an appropriately small diameter and/or through the use of hydrophilic and/or hydrophobic materials or coatings. Substrate <NUM> may further comprise an alphanumeric <NUM>, a barcode <NUM>, and/or similar symbol for identification or processing purposes. Referring to <FIG>, sample holder <NUM> may further comprise an enclosure or case for protecting or sealing substrate <NUM> and the biological samples contained in through-holes <NUM>. The case may comprise a base <NUM> and a cover <NUM> that are configured to seal substrate <NUM> between base <NUM> and cover <NUM>, for example, to reduce or prevent evaporation of the biological samples. Cover <NUM> is made of a transmissive material and comprises a top surface <NUM> and an opposing bottom surface <NUM> for providing optical access to substrate <NUM>. One or both surfaces <NUM>, <NUM> may comprise an antireflective coating, for example, to reduce retro-reflections of light from excitation beam <NUM> back toward optical sensor <NUM>. Additionally or alternatively, one or both surfaces <NUM>, <NUM> may be disposed at an angle relative to a front surface of substrate <NUM>, for example, to reduce retro-reflections of light from excitation beam <NUM> back toward optical sensor <NUM>. Referring to <FIG>, one or more sample holders <NUM> may be retained by or mounted on a carrier <NUM> that is configured to be received by sample processing system <NUM>. In the illustrated embodiment shown in <FIG>, carrier <NUM> is configured to retain four or less sample holders <NUM>. For clarity, not all the through-holes.

In the illustrated embodiment shown in <FIG>, each through-hole <NUM> has a diameter of about <NUM> micrometers, a thickness of or about <NUM> micrometer, and a volume of or about <NUM> nanoliters. Through-holes <NUM> have a nominal spacing in the illustrated embodiment of about <NUM> micrometers center to center. As discussed in greater detail below, optical system <NUM> may be configured to allow imaging and processing of biological samples contained in through-holes having in this range. Additionally or alternatively, system <NUM> and/or optical system <NUM> is configured receive and process a sample holder <NUM> having smaller through-hole diameter and/or a smaller nominal spacing than in the illustrated embodiment of <FIG>. For example, optical system <NUM> may be configured to allow system <NUM> to receive and process a sample holder <NUM> comprising through-holes having a diameter that is less than or equal to <NUM> micrometer and/or a volume that is less than or equal to <NUM> nanoliters. Alternatively, optical system <NUM> may be configured to allow system <NUM> to receive and process a sample holder <NUM> comprising through-holes having a diameter that is less than or equal to <NUM> micrometer and/or a volume that is less than or equal to one nanoliter. In certain embodiments, an initial sample or solution for a sample holder, such as sample holders <NUM>, <NUM>, may be divided into hundreds, thousands, tens of thousands, hundreds of thousands, or even millions of reaction sites, each having a volume of, for example, a few nanoliters, about one nanoliter, or less than one nanoliter (e.g., <NUM>'s or <NUM>'s of picoliters or less).

In the illustrated embodiments shown in <FIG>, sample holders <NUM>, <NUM> have a rectangular shape; however, other shapes may be used, such as a square or circular shape. In certain embodiments, a sample holder such as sample holder <NUM> has a square shape and an overall dimension of <NUM> millimeter by <NUM> millimeter. In such embodiments, the sample holder may have an active area, region, or zone with a dimension of <NUM> millimeter by <NUM> millimeter. As used herein, the terms "active area", "active region", or "active zone" mean a surface area, region, or zone of a sample holder, such as the sample holders <NUM> or <NUM>, over which reaction regions, through-holes, or solution volumes are contained or distributed. In certain embodiments, the active area of sample holder <NUM> may be increased to <NUM> millimeter by <NUM> millimeter or larger, for example, a <NUM> millimeter by <NUM> millimeter substrate dimension.

In the illustrated embodiment of <FIG>, through-holes <NUM> may have a characteristic diameter of or about <NUM> micrometer and a pitch of or about <NUM> micrometers between adjacent through-holes. In other embodiments, through-holes <NUM> have a characteristic diameter of or about <NUM> micrometer and have a pitch of or about <NUM> micrometers between adjacent through-holes. In yet other embodiments, through-holes <NUM> have a characteristic diameter of that is less than or equal <NUM> micrometers, for example, a characteristic diameter that is less or equal to <NUM> micrometers or less or equal to <NUM> micrometers. In other embodiments, through-holes <NUM> have a characteristic diameter that is less than or equal to <NUM> micrometers, less than or equal to <NUM> micrometers, or less than or equal to one micrometer. The pitch between through-holes may be less than or equal to <NUM> micrometers, for example, less than or equal to <NUM> micrometers, less than or equal to <NUM> micrometers, or less than or equal to <NUM> micrometers.

In certain embodiments, sample holder <NUM> comprises a substrate having a thickness between the opposing surfaces of sample holder <NUM> that is at or about <NUM> micrometer, wherein each through-hole <NUM> may have a volume of <NUM> nanoliter, <NUM> nanoliters, or somewhere between <NUM> nanoliter and <NUM> nanoliters. Alternatively, the volume of each through-holes <NUM> may be less than or equal to one nanoliter, for example, by decreasing the diameter of through-holes <NUM> and/or the thickness of sample holder <NUM> substrate. For example, each through-holes <NUM> may have a volume that is less than or equal to one nanoliter, less than or equal to <NUM> picoliters, less than or equal to <NUM> picoliters, or less than or equal to <NUM> picoliters. In other embodiments, the volume some or all of the through-holes <NUM> is in a range from one nanoliter to <NUM> nanoliters.

In certain embodiments, the density of through-holes <NUM> is at least <NUM> through-holes per square millimeter. Higher densities are also anticipated. For example, a density of through-holes <NUM> may be greater than or equal to <NUM> through-holes per square millimeter, greater than or equal to <NUM> through-holes per square millimeter, greater than or equal to <NUM> through-holes per square millimeter, greater than or equal to <NUM>,<NUM> through-holes per square millimeter, or greater than or equal to <NUM>,<NUM> through-holes per square millimeter.

Advantageously, all the through-holes <NUM> with an active area may be simultaneously imaged and analyzed by an optical system. In certain embodiments, active area comprises over <NUM>,<NUM> through-holes <NUM>. In other embodiments, active area comprises at least <NUM>,<NUM>, at least <NUM>,<NUM>, at least <NUM>,<NUM>, or at least <NUM>,<NUM>,<NUM> through-holes.

In certain embodiments, through-holes <NUM> comprise a first plurality of the through-holes characterized by a first characteristic diameter, thickness, or volume and a second plurality of the through-holes characterized by a second characteristic diameter, thickness, or volume that is different than the first characteristic diameter, thickness, or volume. Such variation in through-hole size or dimension may be used, for example, to simultaneously analyze two or more different nucleotide sequences that may have different concentrations. Additionally or alternatively, a variation in through-hole <NUM> size on a single substrate <NUM> may be used to increase the dynamic range of a process or experiment. For example, sample holder <NUM> may comprise two or more subarrays of through-holes <NUM>, where each group is characterized by a diameter or thickness that is different a diameter or thickness of the through-holes <NUM> of the other or remaining group(s). Each group may be sized to provide a different dynamic range of number count of a target polynucleotide. The subarrays may be located on different parts of substrate <NUM> or may be interspersed so that two or more subarrays extend over the entire active area of sample holder <NUM> or over a common portion of active area of sample holder <NUM>.

In certain embodiments, at least some of the through-holes <NUM> are tapered or chamfered over all or a portion of their walls. The use of a chamfer and/or a tapered through-holes have been found to reduce the average distance or total area between adjacent through-holes <NUM>, without exceeding optical limitations for minimum spacing between solution sites or test samples. This results in a reduction in the amount liquid solution that is left behind on a surface of substrate <NUM> during a loading process. Thus, higher loading efficiency may be obtained, while still maintaining a larger effective spacing between adjacent solution sites or test samples for the optical system.

The system (<NUM>) is configured to receive and process at least two different sample holders <NUM> having different numbers of sample cells (wells <NUM>). Thus, system <NUM> may be configured to receive and process sample holders <NUM> containing <NUM> samples and sample holders <NUM> containing <NUM> wells and/or <NUM> well or/or more than <NUM> wells. Additionally or alternatively, system <NUM> may be configured to receive and process different sample formats or container configurations. For examples, in addition to receiving a sample holder <NUM> comprising a predetermined number of wells, system <NUM> may also be configured to receive and process one or more sample holders <NUM> comprising the plurality of through-holes <NUM>. In certain embodiments, system <NUM> is configured to receive and process four different types of sample holders. Some of the characteristics of wells or through-holes used in these four sample holders are listed in Table <NUM> below.

Referring again to <FIG>, optical sensor <NUM> may comprise one or more photodetectors or photosensors, for example, one or more photodiodes, photomultiplier tubes, or the like. Alternatively, optical sensor <NUM> may comprise a one-dimensional or two-dimensional photodetector array <NUM>, such as a charge-coupled device (CCD), complementary metal-oxide-semiconductor (CMOS), or the like. In the illustrated embodiment in <FIG>, photodetector array <NUM> comprises a two dimensional array of photosensitive pixels defining photosensitive surface upon which an optical image or signal may be formed by emission optical system <NUM>.

Excitation source <NUM> may be an excitation light source the produces electromagnetic radiation that is primarily or exclusively within the visible waveband of the electromagnetic spectrum. Excitation source <NUM> may be a halogen lamp, a Xenon lamp, high-intensity discharge (HID) lamp, one or more light emitting diodes (LEDs), one or more laser, or the like. In certain embodiments, excitation source <NUM> comprises a plurality of light sources having different emission wavelength ranges to excite different fluorescent dyes in biological samples <NUM>, for example, a plurality of LED light sources having different colors or emission wavelength ranges. In such embodiments, excitation filter assembly <NUM> may be omitted or may be incorporated for use with at least some of the different light sources to further limit the wavelength range of light or radiation reaching samples <NUM>.

In certain embodiments, excitation source <NUM> comprises one or more broadband or white light LED sources. For example, excitation source <NUM> may comprise a high power, broadband source having at least <NUM> watts of total output optical power, at least <NUM> watts of output optical power, or at least <NUM> watts of output optical power. In such embodiments, excitation filter assembly <NUM> may be incorporated to limit or define the spectral content of the radiation or light received by samples <NUM> and/or sample holder <NUM>, <NUM>. The spectral content of the broadband source <NUM> may be configured to favorably provide more energy over wavelength ranges that, for example, correspond to probes or dye molecules in samples <NUM> that are less efficient, are typically found lower concentrations, or otherwise require more photonic energy that other dyes contained in samples <NUM>.

In a non-limiting example, in certain embodiments, excitation source <NUM> comprises a single broadband LED having a total optical power of greater than <NUM> watts over the spectral range produced by the LED. The spectral output characteristics of such an excitation source are shown by the solid line in the graphs shown in <FIG>. The horizontal axis corresponds to the wavelength of radiation emitted by the LED excitation source <NUM>, while the vertical axis is relative output intensity. The "relative intensity" for the plot in <FIG> is a percentage value that is defined as <NUM> times the intensity measured at a given wavelength divided by the maximum intensity measured at any wavelength within range of wavelengths produced by the LED. For example, according to the plot in <FIG>, the measured intensity out of the LED at a wavelength of <NUM> nanometer is about <NUM> percent of the maximum intensity, where the maximum measured intensity occurs at an output wavelength of <NUM> nanometers. By way of comparison, similar data for a halogen lamp used in a prior art system is also shown in <FIG> as dashed lines. The sets of double lines with numeral in between indicate the approximate transmission wavelength bands for the excitation filters shown in <FIG>.

For the illustrated embodiment shown in Table <NUM>, the characteristic cell diameter and volume of sample holder D is much smaller than that of sample holders A-C. As a result, a typical fluorescence signal produced by sample holder D is much smaller than a typical fluorescence signal produced by sample holders A-C under similar conditions, for example, when using biological samples containing similar concentrations of a biological test sample and/or a fluorescent probe or reference dye. For these reasons, the halogen excitation source shown in <FIG> may not provide sufficient intensity or power density for fluorescent probes or dyes excited by light in the wavelength range provided by filters <NUM>-<NUM> in <FIG>.

In certain embodiments, fluorescent probes or dyes excited by light in the wavelength ranges provided by excitation filters <NUM>, <NUM>, and <NUM> in <FIG> are either more commonly used or are of greater importance than those excited by light in the wavelength range provided by filters <NUM>, <NUM>, and/or <NUM>, for example, in the wavelength range provided by either filters <NUM> or <NUM>. For example, in certain embodiments, the dyes FAM™ (fluorescein amidite), VIC®, and ROX™ are used, which dyes are commercially available from Life Technologies in Carlsbad, CA. In such embodiments, excitation filter <NUM> is used to excite the dye FAM™, excitation filter <NUM> may be used to excite the dye VIC®, and excitation filter <NUM> may be used to excite the dye ROX™. Additionally or alternatively, it may be that fluorescent probes or dyes excited by light in the wavelength ranges provided by filters <NUM>, <NUM>, and/or <NUM> are not used with all types of sample holders A-D and/or with all types of sample holders <NUM>, <NUM>, for example, are not used with sample holders D and/or sample holder <NUM>. In such embodiments as these, an excitation source <NUM> comprising an LED source having spectral characteristics the same or similar to those shown in <FIG> has an unexpected beneficially measurable or useful signal from all or most filters <NUM>-<NUM> for at least some sample holders <NUM>, <NUM> (e.g., for all sample holders except sample holder D in Table <NUM>), even though (<NUM>) the spectral power or intensity of the LED source for fluorescent probes or dyes excited by light in the wavelength range provided by filters <NUM> and <NUM> is less than that for the halogen source shown in <FIG>, and (<FIG>) the spectral power or intensity of the LED source for fluorescent probes or dyes excited by light in the wavelength range provided by filters <NUM> and <NUM> is less than that for fluorescent probes or dyes excited by light in the wavelength range provided by filters <NUM>, <NUM>, and/or <NUM>. It has been discovered that, due to the relatively large sample volumes provided by the sample cells in sample holders A-C in Table <NUM>, an LED source such as that characterized in <FIG> is able to provide enough excitation energy to the biological samples so that system <NUM> is able to process the signals or images received by optical sensor <NUM>.

Accordingly, it has been discovered that instrument or system <NUM> can process biological samples to provide useful data using a broadband LED that produces light or radiation having a maximum intensity and/or power density at a wavelength that is less than <NUM> nanometers and/or that is less than <NUM> nanometers. For example, instrument or system <NUM> can provide useful PCR data (e.g., qPCR and/or dPCR data) using such a broadband LED, such as that represented in <FIG>. The result is an instrument that can provide data, such as PCR data, over a wide range of sample sizes and sample holder or cell formats, for example, all the sample sizes and sample cell formats listed in Table <NUM>.

In certain embodiments, system <NUM> includes an excitation source <NUM> comprising an LED having a spectral profile characterized by a maximum intensity or output power at a wavelength that is less than a first predetermined wavelength or wavelength range, and an intensity or output power that is less than <NUM> percent the maximum value at a second wavelength or wavelength range. For example, system <NUM> may include an excitation source <NUM> comprising an LED having a spectral profile characterized by a maximum intensity or output power at a wavelength that is less than <NUM> or <NUM> nanometers and an intensity or output power that is less than <NUM> percent the maximum value at a wavelength of <NUM> nanometer and/or <NUM> nanometers. In other embodiments, system <NUM> includes an excitation source <NUM> comprising an LED having a spectral profile characterized by a maximum intensity or output power at a wavelength that is less than <NUM> or <NUM> nanometers and an intensity or output power that is less than <NUM> percent or less than <NUM> percent the maximum value at a wavelength of <NUM> nanometer and/or <NUM> nanometers. In certain embodiments, the system <NUM> further comprise an emission optical system <NUM> that is able to provide useful biological data (e.g., PCR data) for sample cells having a diameter of less than <NUM> micrometer, less than <NUM> micrometers, or less than <NUM> micrometers that contain fluorescent probes or dye molecule that fluoresce at excitation wavelengths that are less than or equal to <NUM> nanometer, while also being able to provide useful biological data (e.g., PCR data) for sample cells having a diameter of greater than <NUM> millimeters or greater than <NUM> millimeters that contain fluorescent probes or dye molecule that fluoresce at excitation wavelengths that are greater than or equal to <NUM> nanometer or greater than or equal to <NUM> nanometers.

The system may comprise all or portions of the system <NUM> shown in <FIG>, or a similar such system. The excitation source may be characterized by a spectral function of optical output power or intensity of the excitation source verses wavelength of output power or intensity. The spectral function may be characterized by (a) a minima wavelength corresponding to a local minima value of the optical output power or intensity of the excitation source, (b) a first maxima wavelength corresponding to a first local maxima of optical output power or intensity of the excitation source, and (c) a second maxima wavelength corresponding to a second local maxima of optical output power or intensity of the excitation source. The optical output power or intensity at the first local maxima is greater than the optical output power or intensity at any wavelength less than the minima wavelength, while the optical output power or intensity at the second local maxima is greater than the optical output at any wavelength greater than the minima wavelength. The minima wavelength has a value that is between the first maxima wavelength and the second maxima wavelength. In certain embodiments, the optical output power or intensity is a relative intensity of the excitation source, a relative power of the excitation source, a relative luminous flux of the excitation source, or a radiant flux of the excitation source. For example, referring again to <FIG> as an example, a spectral function according to the above embodiment is illustrated in which (a) the minima wavelength is at or about <NUM> nanometers and corresponds to a relative intensity of about <NUM> units, (b) the first maxima wavelength is at or about <NUM> nanometers and corresponds to a relative intensity of about <NUM> units, and (c) the second maxima wavelength is at or about <NUM> nanometers and corresponds to a relative intensity of about <NUM> units.

When used in system <NUM> according to embodiments of the present invention, another unexpected benefit of an LED excitation source <NUM> as described in the previous paragraph and/or as illustrated in <FIG> is that infrared (IR) emissions from excitation source <NUM> are much lower than, for example, a halogen light source like or similar to that shown in <FIG>. Thus, embodiments of the present invention provide reduced IR noise without the need extra optical element such as so-called "hot mirrors" to block IR emissions.

The output intensity, power, or energy of excitation source <NUM> may also be varied depending on a condition or variable value, for example, experiment or run conditions of system or instrument <NUM>, experiment or run conditions of optical system <NUM>, experiment or run conditions of sample processing system <NUM>, or the like. For example, excitation source <NUM> may be an LED light source in which the output intensity, power, or energy is varied depending on one or more of the conditions and/or variable values. In such embodiments, the output intensity, power, or energy of the LED may be varied by adjusting or changing a current or voltage driving the LED, and/or by adjusting or changing a duty cycle of the LED. The output intensity, power, or energy of excitation source <NUM> is changed depending on the type of sample holder being used in system <NUM>. For example, in certain embodiments, excitation source <NUM> may be an LED that is run at full output power, intensity, or energy - or at a higher power setting output power, intensity, or energy - when sample holder D from Table <NUM> is used. By contrast, the LED may be run at a lower output power, intensity, or energy when a different sample holder is used, for example, sample holder A, B, or C, from Table <NUM> is used. Such an arrangement allows system <NUM> to provide emission data for the smaller sample volume sizes and/or lower sample concentrations that occur when sample holder A is used, while also avoiding a saturation of optical sensor <NUM> when other larger sample volumes and/or higher sample concentrations are used.

Referring again to <FIG>, lens <NUM> is configured to form an image on photodetector array <NUM>, for example, by focusing collimated radiation entering from a particular direction to a spot or point, for example, to a diffraction limited spot or a nearly diffraction limited spot. Lens <NUM> may be a simple lens, such as a plano-convex lens, plano-concave lens, bi-convex lens, bi-concave lens, meniscus lens, or the like. Alternatively, lens <NUM> may comprise a compound lens such as a doublet lens or triplet lens that may, for example, comprise different lens materials selected to correct or reduce a chromatic aberration. In other embodiments, lens <NUM> comprises system of lenses such as a camera lens system or microscope objective, for example, a commercially available camera lens. The camera lens system may be a commercially available camera lens comprising a conventional lens system design, for example, a double Gauss design, a Cooke triplet design, retrofocus lens design (e.g., Distagon lens design), a Tessar lens design, or the like.

Lens <NUM> may be a single field lens, for example, configured to provide a telecentric optical system when combined with the remaining optical elements of excitation optical system <NUM> and/or emission optical system <NUM>. In such embodiments, lens <NUM> may be a simple lens, such as a plano-convex lens, plano-concave lens, bi-convex lens, bi-concave lens, meniscus lens, or the like. Alternatively, lens <NUM> may comprise a doublet lens or triplet lens, for example, comprising different lens material to correct for a chromatic aberration. Additionally or alternatively, lens <NUM> may comprise a Fresnel lens or a diffractive optical element, surface, or pattern. In certain embodiments, lens <NUM> may comprise a lens system, for example, a field lens in combination with an additional lens or lenslet array configured to focus light within a sample well of sample holder <NUM>. The lenslet array may comprise a Fresnel lens or a diffractive optical element, surface, or pattern. Examples of such lens configurations are also described in USPN <NUM>,<NUM>,<NUM>.

Referring to <FIG>, in certain embodiments, heated lid <NUM> comprises a lenslet array <NUM>, for example, for use with sample holder <NUM> (e.g., like sample holders A-C listed in Table <NUM>) for focusing light from excitation beam <NUM> into a well or cavity of a sample holder, such as sample holders <NUM> in the illustrated embodiment. With additional reference to <FIG>, heated lid <NUM> may additionally or alternatively comprise an optical window <NUM> for providing thermal isolation or improved thermal performance of the thermal environment in or around a sample holder, such as sample holder <NUM> in the illustrated embodiment. In certain embodiments, convective current can be produced when window <NUM> is not located as shown in <FIG>. Such convective heat flow has been found to result in higher temperature or thermal non-uniformity (TNU) in substrate <NUM>, in sample holder <NUM>, and/or between samples <NUM> than may be acceptable in some applications. Accordingly, placement of window <NUM> between lens <NUM> and sample holder <NUM> can decrease the amount of convective currents around sample holder <NUM> and lead to a decreases in TNU.

Optical window <NUM> may be used in addition to or in place of optical window <NUM> shown in <FIG>. Either or both windows <NUM>, <NUM> may be disposed parallel to a surface of sample holder <NUM> and/or perpendicular to optical axis <NUM>. Alternatively, one or both windows <NUM>, <NUM> may be disposed at an angle relative to a surface of sample holder <NUM> and/or at an acute angle to optical axis <NUM>, for example, to reduce retro-reflections of light from excitation beam <NUM> back toward optical sensor <NUM>. One or both windows <NUM>, <NUM> may comprise an antireflective coating to reduce retro-reflections of light from excitation beam <NUM> back toward optical sensor <NUM>. The antireflective coating may be used in addition to, or as an alternative to, tilting one or both windows <NUM>, <NUM>. Thus, system <NUM> is able to accommodate and provide useful biological data (e.g., PCR data) for sample holders having a diversity of optical requirements by providing heated lids <NUM> having different optical characteristics from one another and/or serving differing thermal requirements for sample holders, such as sample holders <NUM>, <NUM> (e.g., sample holders A-D listed in Table <NUM>).

In certain embodiments, the combination of lenses or lens systems <NUM>, <NUM> is selected to provide a predetermined optical result or image quality. For example, in order to reduce system cost or to simplify the emission optical system <NUM> design, lens <NUM> may comprise a commercially available camera lens. Such lenses can provide very high image quality (e.g., images with low chromatic and monochromatic aberration) under certain viewing conditions. However, the careful balance of higher order aberrations incorporated into such camera lens design used to provide such high image quality can be disturbed with the introduction of other lenses into an imaging system. For example, in the illustrated embodiment shown in <FIG>, a field lens such as lens <NUM> is added to emission optical system <NUM>. Lens <NUM> is common to both excitation optical system <NUM> and emission optical system <NUM> to provide both a generally more compact optical system and efficient transfer of fluorescent energy from a sample to the detection system.

In prior art systems, a field lens having a plano-convex lens shape or figure has been found to provide certain favorable characteristic in this respect, for example, to provide a telecentric lens system configured to provide even illumination over a large field of view. However, to provide an acceptably low level of optical aberrations, such prior art systems also incorporate a custom camera lens design in order to reduce overall system aberrations when used in combination the plano-convex field lens. In particular, due to the extended field of view used to simultaneously image a large number of biological samples, the camera lens was designed to provide low amounts of field curvature. However, it has been discovered that the combination of a plano-convex lens with a conventional or commercially available camera lens can result in large amounts of field curvature that are undesirable. It has been further discovered that field curvature can be significantly reduced by combining a biconvex field lens <NUM> with a conventional or commercially available camera lens, as illustrated in <FIG>. This result is surprising, since a biconvex lens would normally be expected to reduce overall image quality in a telecentric lens system. For example, it has been found that when lens <NUM> comprises a commercially available camera lens of a retrofocus design (e.g., a Distagon lens design), the amount of field curvature produced when field lens <NUM> is a biconvex lens is much smaller than the amount of field curvature produced when field lens <NUM> is a plano-convex lens.

Emission filter assembly <NUM> may comprise a first filter module <NUM> characterized by a first optical power and a first filter <NUM> having a first filter function or transmission range 140a. In the illustrated embodiment, first filter function 140a is shown as filter number <NUM> in the table of <FIG> and is characterized by a first low-pass wavelength <NUM> of <NUM> nanometers and a first high-pass wavelength <NUM> of <NUM>, so that light within this wavelength range is transmitted, or largely transmitted, through the first filter <NUM>, while light or other electromagnetic radiation outside this wavelength range is blocked, or substantially blocked, by first filter <NUM>. The wavelengths listed in <FIG> may represent the wavelengths at which the transmission of a filter is one-half the maximum transmission of the filter over the transmission wavelength range. In such cases, the difference between high-pass wavelength and the low-pass wavelength define a full width at half maximum transmission (FWHM) range.

Emission filter assembly <NUM> also includes a second, and optionally a third, filter component, element, or module <NUM>, <NUM>. Second and third filter modules142, <NUM> are characterized by second and third filters <NUM>, <NUM> having a second and third filter functions or transmission ranges 145a, 146a. Either or both filter modules <NUM>, <NUM> may have an optical power that is the same as, or different from, the optical power of first filter module <NUM>. At least one of the filter modules <NUM>, <NUM> may have an optical power of zero, which power may in general be either positive or negative. Filter functions 145a, 146a comprise second and third low-pass wavelengths <NUM>, <NUM> second and third high-pass wavelengths <NUM>, <NUM>, respectively, for example, as filter numbers <NUM> and <NUM> in the table of <FIG>. Second and third low-pass wavelengths <NUM>, <NUM> may be different than the low-pass wavelength <NUM> and/or may be different from one another. Similarly, second and third high-pass wavelengths <NUM>, <NUM> may be different than the high-pass wavelength <NUM> and/or may be different from one another. In the illustrated embodiment, the transmission wavelength bands for filters <NUM>, <NUM>, <NUM> (wavelengths <NUM> to <NUM>, <NUM> to <NUM>, and <NUM> to <NUM>) do not overlap; however, in other embodiments, there may be at least some overlap in the wavelength bands between two or more of the filters in emission filter assembly <NUM>. In certain embodiments, one or more of functions 140a, 145a, 146a may comprise a function that is different than the simple bandpass configuration illustrated in <FIG>.

<FIG> illustrates various filters available for use with excitation filter assembly <NUM>. In <FIG>, excitation filter assembly <NUM> comprises three filters, for example, filters <NUM>, <NUM>, and <NUM> in <FIG>, which in use may correspond to filters <NUM>, <NUM>, and <NUM> shown in <FIG> for emission filter assembly <NUM>. At least some of the filter modules <NUM> of excitation filter assembly <NUM> may have non-zero optical powers, which power may in general be either positive or negative. Alternatively, all the filter modules <NUM> may have zero or about zero optical power. In certain embodiments, selection of a particular filter module <NUM> is associated with a particular filter module <NUM> of filter assembly <NUM>. Alternatively, filter modules <NUM>, <NUM> may be selected independently of one another.

In the illustrated embodiment shown in <FIG>, only three filter modules are shown for each filter assembly <NUM>, <NUM>; however, either or both filter assemblies may comprise more or less than three filter modules. For example, <FIG> each show a total of <NUM> filters, each of which filters may be associated an optical power (not shown). In certain embodiment, either or both filter assemblies <NUM>, <NUM> contain all six filters shown in the table shown in <FIG>, respectively. Alternatively, either or both filter assemblies <NUM>, <NUM> may comprise less than six filters.

Filter functions 145a, 146a comprise respective second low-pass wavelengths <NUM>, <NUM> that may be different than the first low-pass wavelength <NUM> and may be different from one another. Each filter of the filters in emission filter assembly <NUM> or in excitation filter assembly <NUM> may comprise a transmission range of electromagnetic radiation or light that is different and non-overlapping from the remaining filters of filter assembly <NUM> or filter assembly <NUM>. Alternatively, two or more of the filters in filter assembly <NUM> or in filter assembly <NUM> may comprise transmission ranges of electromagnetic radiation or light that at least partially overlap one another.

In certain embodiments, the optical power one more or more of filter modules <NUM>, or of each filter modules <NUM>, is selected to compensate for or reduce an optical aberration of the remaining optical elements of emission optical system <NUM> or excitation optical system <NUM> over a wavelength range of the filter being used. For example, in order to provide a predetermined image resolution or quality for various of the filter modules <NUM> at an image plane of optical sensor <NUM> or emission optical system <NUM>, the optical powers of some or all of filter modules <NUM> may be selected to compensate for or reduce a chromatic or sphero-chromatic aberration introduced by emission optical system <NUM> over different filter wavelength ranges. Additionally or alternatively, one or more of filter modules <NUM> or of filter modules <NUM> may comprise a monochromatic aberration, such as spherical aberration, astigmatism, or coma, that is configured to alter, adjust, or reduce an overall aberration of emission optical system <NUM> or excitation optical system <NUM>.

In certain embodiments, the optical power or a monochromatic aberration of one or more of filter modules <NUM> is configured to at least partially correct or adjust an image or focus of sample holder <NUM> and/or of at least some of the biological samples <NUM> in an image plane at or near a detection surface of optical sensor <NUM>. For example, in the illustrated embodiment, the optical powers of filter modules <NUM>, <NUM>, <NUM> are all different from one another, with third filter module <NUM> having an optical power of zero or about zero. The optical power of filter modules <NUM>, <NUM> may be selected so that an effective focal length of emission optical system <NUM> is adjusted over the transmission wavelength range of each filter <NUM>, <NUM> is the same or about the same as the effective focal length when filter <NUM> is located in the emission optical system <NUM>. Additionally or alternatively, the optical power of filter modules <NUM>, <NUM> may be selected so that the image quality produced when corresponding filter <NUM>, <NUM> is inserted into emission optical system <NUM> is the same or similar to the image quality produced when filter <NUM> is inserted into emission optical system <NUM>. For example, the optical power for each filter module <NUM> may be selected so that images of biological samples <NUM> are the same size, or about the same size, for each filter module <NUM>, <NUM>, <NUM>. Additionally or alternatively, the optical power for each filter module <NUM> may be selected so that a magnification and/or aberration of images of biological samples <NUM> are the same, or about the same, for each filter module <NUM>. In certain embodiments, two or more of the optical powers may be the equal to one another. In general filter modules <NUM> and/or <NUM> may have optical powers that are greater than zero or less than zero in order to provide a desired correction or adjustment to the emission optical system <NUM> and/or images produced therefrom.

Beamsplitter <NUM> may be configured to selectively reflect a large amount of emitted light or radiation from excitation source <NUM> that is transmitted through a selected excitation filter module <NUM> and then directed toward sample holder <NUM>, <NUM>. For example, the coated beamsplitter <NUM> may comprise a dichroic reflector that is configured to reflect at least <NUM> percent or at least <NUM> percent of incident light transmitted through excitation filter module <NUM>. The same coating for beamsplitter <NUM> can additionally be configured to transmit a large amount of emission light or radiation from biological samples <NUM>, for example, to transmit at least <NUM> percent or at least <NUM> percent of light or radiation emitted by biological samples <NUM>. In certain embodiments, a different beamsplitter <NUM> is associated with each different filter module <NUM>, for example, by attaching the different beamsplitters <NUM> to excitation filter assembly <NUM>. In certain embodiments, only some of the beamsplitters <NUM> are wavelength selective or dichroic beamsplitters, while others of beamsplitters <NUM> associated with some of excitation filter modules <NUM> are not wavelength selective, for example, a <NUM>/<NUM> beamsplitter that reflect <NUM> percent of incident radiation over a broad band of wavelengths. In such embodiments, excitation light or radiation not reflected by a beamsplitter <NUM>, but transmitted through the beamsplitter <NUM>, may be intercepted by an emission filter module <NUM> and directed to optical sensor <NUM> in the form of noise.

In certain embodiments, optical system <NUM> comprises a plurality of optical modules, where each optical module comprises a beamsplitter <NUM> and an excitation filter or filter module <NUM> and/or an emission filter or filter module <NUM>. Each optical module may be inserted or removed from excitation optical path <NUM> and/or emission optical path <NUM>. In some embodiments, each module comprises a beamsplitter <NUM> that is commonly mounted with one of the excitation filters or filter modules <NUM>. In such embodiments, a beamsplitter/excitation filter pair <NUM>, <NUM> may be inserted and removed from excitation optical path <NUM>, while emission filters or filter modules <NUM> may be inserted and removed from emission optical beam path <NUM> independently of the beamsplitter/excitation filter pairs <NUM>, <NUM>.

In certain embodiments, noise from excitation light or radiation transmitted through a beamsplitter <NUM> is reduced by reducing the size of the corresponding emission filter module <NUM>. However, the size reduction of the corresponding emission filter module <NUM> may be limited so as to avoid loss of signal from at least some of the biological samples <NUM>, <NUM>, for example, due to vignetting effects on the more peripherally located samples. It has been discovered that a reduction in excitation radiation noise can be accomplished without significant loss of emission radiation signal by configuring the emission filters to have a shape that is the same as, or similar to, the shape of the area of sample holder <NUM>, <NUM> containing samples <NUM>, <NUM>. For example, it can be seen in <FIG>, or <FIG> that a rectangular area is defined by an active area over which one or more of sample holders <NUM>, <NUM> contain samples or sample cells within the field of view of optical sensor <NUM>. In such cases, it has been found that a rectangular shaped emission filter <NUM>, <NUM>, <NUM> or emission filter module <NUM>, <NUM>, <NUM> provides reduced noise from excitation radiation transmitted through beamsplitter <NUM>, without a significant loss of emission signal from samples <NUM>, <NUM> or uneven illumination from samples over the entire area of sample holders <NUM>, <NUM>. In certain embodiments, the rectangular emission filter <NUM>, <NUM>, <NUM> or emission filter module <NUM>, <NUM>, <NUM> has the same, or a similar, aspect ratio as that defined by an active area of sample holders <NUM>, <NUM>, by carriers <NUM>, <NUM>, or by area of samples <NUM>, <NUM> that are within the field of view or field of regard of optical sensor <NUM>. For example, the aspect ratio of a rectangular emission filter (e.g., filter <NUM>, <NUM>, and/or <NUM>) or filter module (e.g., filter module <NUM>, <NUM>, or <NUM>) may be selected to be within <NUM> percent, <NUM> percent, <NUM> percent, or <NUM> percent of the aspect ratio of the active area of a sample holder (e.g., sample holders <NUM> or <NUM>) or of a group of sample holders (e.g., the four sample holders <NUM> shown in <FIG>).

During operation, biological samples <NUM> are disposed in a sample holder, for example in sample holder <NUM>, sample holder <NUM>, or the like. Biological samples <NUM> may include one or more nucleotide sequences, amino acid sequences, or other biological macromolecules including, but not limited to, oligonucleotides, genes, DNA sequences, RNA sequences, polypeptides, proteins, enzymes, or the like. In addition, biological samples <NUM> may include other molecules for controlling or monitoring a biological reaction including, but not limited to, primers, hybridization probes, reporter probes, quencher molecules, molecular beacons, fluorescent dyes, chemical buffers, enzymes, detergents, or the like. Additionally or alternatively, biological samples <NUM> may include one or more genomes, cells, cellular nucleuses, or the like.

Once the biological samples are loaded, one or more sample holders are loaded or mounted within system <NUM>. In the illustrated embodiment shown in <FIG>, one or more sample holders are mounted in to carrier <NUM> or <NUM>, which in turn is received by block <NUM> system <NUM> and may be subsequently covered or secured by heated lid <NUM>. As discussed above herein, block <NUM> and heated lid <NUM> may be removably mounted or secured within system <NUM>, for example, so either or both may be exchanged for another block or heated lid that is configured for use with a particular sample holder or carrier. Once the sample holder has been received by sample processing system <NUM>, optical system <NUM> is used to monitor or measure one or more biological reactions or processes.

Emission optical system <NUM> of optical system <NUM> comprises an optical axis <NUM>. A first emission beam <NUM> of emission beams <NUM> is emitted by a first biological sample located at or near optical axis <NUM>. First emission beam <NUM> passes through emission optical system <NUM> such that at least a portion of the electromagnetic radiation from the sample produces a first sample image <NUM> at or near photodetector array <NUM> that is on or near optical axis <NUM>. A second emission beam <NUM> of emission beams <NUM> is simultaneously emitted by second biological sample located at or near an outer edge location of the array of biological samples <NUM>. Second emission beam <NUM> also passes through emission optical system <NUM> such that at least a portion of the electromagnetic radiation from the sample produces a second sample image <NUM> at or near optical sensor <NUM> that is displace from optical axis <NUM>. Emission beams <NUM>, <NUM> may be fluorescence beams produced by different probe molecules contained in the two respective samples in response to excitation beam <NUM>. Depending upon the particular excitation filter module <NUM> selected, emission beams <NUM>, <NUM> have a wavelength or wavelength range corresponding to the particular probe molecule that is excited by radiation from excitation beam <NUM> that is transmitted by the selected excitation filter module <NUM>. For example, when filter number <NUM> in <FIG> may be used to filter radiation from excitation beam <NUM> and used in combination with emission filter number <NUM> in <FIG> (filter <NUM> of filter module <NUM> in <FIG>) to transmit radiation from emission beams <NUM>, <NUM> onto photodetector array <NUM>. As discussed above herein, the combination of lenses <NUM>, <NUM> may be selected to form images from emission beams <NUM>, <NUM> that is low in monochromatic aberrations, and in particular has a low amount of field curvature. A lateral distance (e.g., in a direction normal to optical axis <NUM>) between the first and second samples may be compared to a lateral distance between the corresponding images produced by emission optical system <NUM> to determine a transverse magnification for the system when filter <NUM> is being used.

In certain embodiments, for radiation within the transmission range of emission filter <NUM>, first and second beams <NUM>, <NUM> are collimated or nearly collimated when they leave lens <NUM> and form images at or near photodetector array <NUM> that have relatively low monochromatic aberrations and define a base system magnification. During use, emission filter assembly <NUM> may be subsequently moved (e.g., translated or rotated) so that emission filter module <NUM> and filter <NUM> are replaced by emission filter module <NUM> and filter <NUM> so the filter <NUM> (filter number <NUM> in <FIG>) now becomes part of the emission optical system <NUM>, as illustrated in <FIG>. Optionally, excitation filter number <NUM> in <FIG> may also be replaced with excitation filter number <NUM> along excitation beam path <NUM>. As a result of chromatic aberrations, for radiation within the transmission range of emission filter <NUM>, first and second beams <NUM>, <NUM> are no collimated, but are divergent when they leave lens <NUM>. Thus, beams <NUM>, <NUM> form images <NUM>, <NUM> at or near photodetector array <NUM> that are further away from a principal plane of lens <NUM> than the images formed when filter <NUM> is present in emission optical system <NUM>. To correct or compensate for this effective change in focal length of emission optical system <NUM> over the transmission wavelength range of filter <NUM>, a lens or optic <NUM> with a net positive optical power is included in filter module <NUM>.

The added optical power to filter module <NUM> and emission optical system <NUM> may be provided by a singlet lens <NUM>, as shown in the illustrated embodiment of <FIG>. The lens may be a plano-convex lens, plano-concave lens, bi-convex lens, bi-concave lens, meniscus lens, or the like. Alternatively, lens <NUM> may comprise a compound lens such as a doublet lens or triplet lens that may, for example, comprise different lens materials selected to correct or reduce a chromatic aberration. Optic <NUM> may additionally or alternatively comprise a diffractive optical element. Optic <NUM> may be either a separate optical element, as shown in <FIG>, or combined with filter <NUM> to form a single element. For example, optic <NUM> and filter <NUM> may be bonded together along a common optical face. Alternatively, optic <NUM> and filter <NUM> be formed together from a single substrate, for example, formed from a filter material having one or both optical surfaces that are curved and/or contain a diffractive optical pattern. In certain embodiments, optic <NUM> is located in a different part of emission optical system <NUM> than shown in <FIG>, for example, on or proximal lens <NUM>, window <NUM>, or beamsplitter <NUM>, or at a location between beamsplitter <NUM> and emission filter assembly <NUM>.

In addition to changing the effective focal length of emission optical system <NUM>, filter <NUM> may also result in a change in transverse magnification for the system. For example, even when lens <NUM> is included in filter module <NUM>, the lateral distance between images <NUM>, <NUM> may be different when filter module <NUM> is used than when filter module <NUM> is used. In addition, the change from filter module <NUM> to filter module <NUM> may introduce or alter various monochromatic aberration of emission optical system <NUM>, for example, a spherical aberration and/or field curvature. Accordingly, optic <NUM> or filter module <NUM> may be configured to at least partially correct or compensate for such differences or changes in magnification and/or in one or more monochromatic aberrations relative to when filter module <NUM> is used. In certain embodiments, system <NUM> or computer system <NUM> may include image processing instructions to at least partially correct or compensate for changes in magnification and/or in one or more monochromatic aberrations introduced by the use of filter module <NUM> into emission optical system <NUM>. The image processing instructions may be used in combination with, or in place of, corrective optic <NUM> to at least partially correct or compensate for changes in produces by the use of filter <NUM> in place of filter <NUM>, including changes in effective system focal length, magnification, chromatic aberrations, and/or one or more monochromatic aberrations such as defocus, spherical aberrations, or field curvature.

In certain embodiments, each filter module <NUM> is disposed, in its turn, along the emission optical path <NUM> at a location where emission beam <NUM>, or some portion thereof, is either diverging or converging, whereby one or more of filter modules <NUM>, <NUM>, <NUM> alters the amount of divergence or convergence to correct or adjust an effective focal length of emission optical system <NUM> and/or a spot size at an image plane of emission optical system <NUM>. In such embodiments, an optical power of at least one of filter modules <NUM>, <NUM>, <NUM> is non-zero (i.e., either positive or negative) over at least the transmission wavelength range or filter function of corresponding filter <NUM>, <NUM>, <NUM>.

In certain embodiments, the optical power of one or more of filter modules <NUM>, or one or more of filter modules <NUM>, is greater than zero and less than one Diopter. For example, the optical power of one or more of filter modules <NUM>, or one or more of filter modules <NUM>, is greater than zero and less than or equal to one-third of one Diopter, less than or equal to one-quarter of one Diopter, or less than or equal to one-eighth of one Diopter. Thus, optical power adjustment, while greater than zero, may be relatively small, so that only sight adjustments are made in the optical characteristics of the emission optical system <NUM> for at least some of the filters <NUM>, <NUM>, <NUM>. Such slight adjustment in optical power in the emission optical system <NUM> for different filters have been found to provide important optical corrections, resulting images created at optical sensor <NUM> that allow for better comparison between image data at different excitation and emission conditions.

While most of the discussion above has related to emission optical system <NUM> and the associated filter module <NUM>, it will be appreciated that embodiments of the present invention also encompass similar treatment, where appropriate, of excitation optical system <NUM> and the associated filter module <NUM>.

In the illustrated embodiment shown in <FIG>, a non-zero optical power for some of filter module <NUM>, <NUM> is provided by a separate lens. Alternatively, a filter module <NUM>, <NUM> may comprise a single optical element having both an optical power and filter transmission function. In certain embodiments, the single optic is made of a single material. Alternatively, two or more materials or elements may be adhered, joined, or bond together to form a filter module. In certain embodiments, the optical power may be provided by a diffractive or holographic optical element or surface. The diffractive or holographic element or surface may be configured to reduce the size or thickness of a filter module. Additionally or alternatively, the diffractive or holographic element or surface may be configured to introduce a chromatic aberration that is used to reduce a chromatic aberration produced by the remaining elements of optical systems <NUM> or <NUM>. In yet other embodiments, one or more of filter assemblies comprises a Fresnel lens or a curved mirror.

In certain embodiments, filter assembly <NUM> and/or <NUM> comprise a carrousel configuration in which different filter modules <NUM> or <NUM> are rotated into and out of the emission optical path <NUM> or excitation optical path <NUM>, respectively. In certain embodiments, filter assembly <NUM> and/or <NUM> comprises interchangeable optical elements having differing optical powers and interchangeable filters having differing filter functions, wherein the optical elements and filters are independently selectable from one another.

First optical element <NUM> is disposed near the optical sensor and is configured to provide images of samples <NUM> and/or sample holder <NUM>. First optic element <NUM> may be a simple lens, such as a plano-convex or bi-convex lens, or a commercially available camera lens, such as a Double Gauss lens, Distagon lens, Angenieux retrofocus lens, Cooke triplet, or the like. In the illustrated embodiment, filter modules <NUM> are located between beamsplitter <NUM> and optical element <NUM>, proximal optical element <NUM>. Second optical element <NUM> may be located near sample holder <NUM> and be configured to provide a telecentric optical system for illumination of the plurality of biological samples <NUM>.

Referring to <FIG>, computer system <NUM> may comprise various detection, data/image processing, and/or control operations or processes may be implemented using hardware, software, firmware, or combinations thereof, as appropriate. For example, some operations or processes may be carried out using processors or other digital circuitry under the control of software, firmware, or hard-wired logic. (The term "logic" herein refers to fixed hardware, programmable logic and/or an appropriate combination thereof, as would be recognized by one skilled in the art to carry out the recited functions. ) Software and firmware can be stored on non-transitory computer-readable media. Additionally or alternatively, at least some operations or processes may be implemented using analog circuitry, as is well known to one of ordinary skill in the art. Additionally, electronic memory or other storage, as well as communication components, may be employed in embodiments of the current invention.

Without limiting the scope of the current invention, the block diagram in <FIG> illustrates one embodiment of a computer system <NUM> for carrying out processing functionality according to various embodiments of system <NUM>. Computer system <NUM> may be utilized to control one or more polymerase chain reaction (PCR), sequencing, and/or genotyping instruments, or the like. Computing system <NUM> can include one or more processors, such as a processor <NUM>. Processor <NUM> can be implemented using a general or special purpose processing engine such as, for example, a microprocessor, controller or other control logic. Computing system <NUM> may include bus <NUM> or other communication medium or mechanism for communicating information, and processor <NUM> coupled with bus <NUM> for processing information.

Further, it should be appreciated that a computing system <NUM> illustrated in <FIG> may be embodied in any of a number of forms, such as a rack-mounted computer, mainframe, supercomputer, server, client, a desktop computer, a laptop computer, a tablet computer, handheld computing device (e.g., PDA, cell phone, smart phone, palmtop, etc.), cluster grid, netbook, embedded systems, or any other type of special or general purpose computing device as may be desirable or appropriate for a given application or environment. Additionally, a computing system <NUM> can include a conventional network system including a client/server environment and one or more database servers, or integration with LIS/LIMS infrastructure. A number of conventional network systems, including a local area network (LAN) or a wide area network (WAN), and including wireless and/or wired components, are known in the art. Additionally, client/server environments, database servers, and networks are well documented in the art. According to various embodiments, computing system <NUM> may be configured to connect to one or more servers in a distributed network. Computing system <NUM> may receive information or updates from the distributed network. Computing system <NUM> may also transmit information to be stored within the distributed network that may be accessed by other clients connected to the distributed network.

Computing system <NUM> also includes a memory <NUM>, which can be a random access memory (RAM) or other dynamic memory, coupled to bus <NUM> for storing instructions to be executed by processor <NUM>. Memory <NUM> also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor <NUM>. Computing system <NUM> further includes a read only memory (ROM) <NUM> or other static storage device coupled to bus <NUM> for storing static information and instructions for processor <NUM>.

Computing system <NUM> may also include a storage device <NUM>, such as a magnetic disk, optical disk, or solid state drive (SSD) is provided and coupled to bus <NUM> for storing information and instructions. Storage device <NUM> may include a media drive and a removable storage interface. A media drive may include a drive or other mechanism to support fixed or removable storage media, such as a hard disk drive, a floppy disk drive, a magnetic tape drive, an optical disk drive, a CD or DVD drive (R or RW), flash drive, or other removable or fixed media drive. As these examples illustrate, the storage media may include a computer-readable storage medium having stored therein particular computer software, instructions, or data. In certain embodiments, storage device <NUM> comprises one or more of memory <NUM> or ROM <NUM>.

Additionally or alternatively, storage device <NUM> may include other similar instrumentalities for allowing computer programs or other instructions or data to be loaded into computing system <NUM>. Such instrumentalities may include, for example, a removable storage unit and an interface, such as a program cartridge and cartridge interface, a removable memory (for example, a flash memory or other removable memory module) and memory slot, and other removable storage units and interfaces that allow software and data to be transferred from the storage device <NUM> to computing system <NUM>.

Computing system <NUM> can also include a communications interface <NUM>. Communications interface <NUM> can be used to allow software and data to be transferred between computing system <NUM> and external devices. Examples of communications interface <NUM> can include a modem, a network interface (such as an Ethernet or other NIC card), a communications port (such as for example, a USB port, a RS-232C serial port), a PCMCIA slot and card, Bluetooth, etc. Software and data transferred via communications interface <NUM> are in the form of signals which can be electronic, electromagnetic, optical or other signals capable of being received by communications interface <NUM>. These signals may be transmitted and received by communications interface <NUM> via a channel such as a wireless medium, wire or cable, fiber optics, or other communications medium. Some examples of a channel include a phone line, a cellular phone link, an RF link, a network interface, a local or wide area network, and other communications channels.

Computing system <NUM> may be coupled via bus <NUM> to a display <NUM>, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device <NUM>, including alphanumeric and other keys, is coupled to bus <NUM> for communicating information and command selections to processor <NUM>, for example. An input device may also be a display, such as an LCD display, configured with touchscreen input capabilities. Another type of user input device is cursor control <NUM>, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor <NUM> and for controlling cursor movement on display <NUM>. A computing system <NUM> provides data processing and provides a level of confidence for such data. Consistent with certain implementations of embodiments of the present teachings, data processing and confidence values are provided by computing system <NUM> in response to processor <NUM> executing one or more sequences of one or more instructions contained in memory <NUM>. Such instructions may be read into memory <NUM> from another computer-readable medium, such as storage device <NUM>. Execution of the sequences of instructions contained in memory <NUM> causes processor <NUM> to perform the process states described herein. Alternatively hard-wired circuitry may be used in place of or in combination with software instructions to implement embodiments of the present teachings. Thus implementations of embodiments of the present teachings are not limited to any specific combination of hardware circuitry and software.

The term "computer-readable medium" and "computer program product" as used herein generally refers to any media that is involved in providing one or more sequences or one or more instructions to processor <NUM> for execution. Such instructions, generally referred to as "computer program code" (which may be grouped in the form of computer programs or other groupings), when executed, enable the computing system <NUM> to perform features or functions of embodiments of the present invention. These and other forms of non-transitory computer-readable media may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, solid state, optical or magnetic disks, such as storage device <NUM>. Volatile media includes dynamic memory, such as memory <NUM>. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus <NUM>.

Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave as described hereinafter, or any other medium from which a computer can read.

Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor <NUM> for execution. For example, the instructions may initially be carried on magnetic disk of a remote computer. A modem local to computing system <NUM> can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus <NUM> can receive the data carried in the infra-red signal and place the data on bus <NUM>. Bus <NUM> carries the data to memory <NUM>, from which processor <NUM> retrieves and executes the instructions. The instructions received by memory <NUM> may optionally be stored on storage device <NUM> either before or after execution by processor <NUM>.

However, it will be apparent that any suitable distribution of functionality between different functional units, processors or domains may be used without detracting from the invention. For example, functionality illustrated to be performed by separate processors or controllers may be performed by the same processor or controller. Hence, references to specific functional units are only to be seen as references to suitable means for providing the described functionality, rather than indicative of a strict logical or physical structure or organization.

Computing system <NUM> may be accessible to an end user through user interface <NUM>, for example, via communications interface <NUM>. Additionally, computer system <NUM> may provide data processing, display and report preparation functions, for example, via display <NUM> and/or one or more input devices <NUM>. All such instrument control functions may be dedicated locally to the system <NUM> and may provide remote control of part or all of the control, analysis, and reporting functions.

Computer system <NUM> may additionally or alternatively, comprise functionality or capabilities for communicating with, and/or controlling, one or more processes, systems, or subsystems of system or instrument <NUM>. For example, computer system <NUM> may comprise one or more interfaces or communications with excitation source <NUM>, excitation source temperature controller <NUM>, sample thermal controller <NUM>, optical system <NUM>, and/or one or more temperature sensors (e.g. an LED temperature sensor <NUM>). The one or more interfaces or communications with optical system <NUM> may include, but is not limited to, optical detector <NUM> (e.g., for adjusting position, controlling gain, frame rate, data collection rate, binning, or the like), filter assemblies <NUM>, <NUM> (e.g. for moving different filters into or out of an excitation or emission beam path), beamsplitter <NUM> (e.g. for moving different beamsplitter into or out of an excitation and/or emission beam path), optical element <NUM> (e.g., for adjusting an image focus and/or lens position), or the like.

Without limiting the scope of the current invention, an instrument <NUM> according to in an exemplary embodiment was constructed that included an excitation source <NUM> that comprised a broad-band LED that produced significant amounts of output power or intensity across a large portion of the visible spectrum. Instrument <NUM> of the exemplary embodiment was also configured to receive and obtain image data from a variety of sample holders <NUM>, <NUM> that included sample holders A, B, C, and D listed in Table <NUM>. Instrument <NUM> of the exemplary embodiment also comprised a computer system <NUM> according to that shown in <FIG>. The optical reader of the instrument <NUM> of the exemplary embodiment was generally arranged in accordance with the system shown in <FIG>. The optical sensor <NUM> was a CCD array detector. A number of instruments <NUM> according to present exemplary embodiment were constructed and operated to obtain data discussed below. Thus, reference to the exemplary embodiment of instrument <NUM> may refer to an individual instrument or the plurality of instruments used in providing resulting instrument design.

The LED <NUM> of the exemplary embodiment was a nominally <NUM> W LED providing approximately <NUM> Lumens of output over the visible waveband. The LED <NUM> had a relative intensity as shown in <FIG>, comprising a maximum power output at a wavelength within the visible range of the electromagnetic spectrum and having a power output that was at least <NUM> percent of the maximum power output over a wavelength range from <NUM> nanometers to <NUM> nanometers. Using filters with the characteristics like those shown in <FIG> and <FIG>, instrument <NUM> was configured to provide the following "channels" for measuring fluorescence levels of at least the dyes shown in Table <NUM>.

LED <NUM> produced sufficient output power to provide qPCR data for channels <NUM>, <NUM>, and <NUM> of Table <NUM> when sample holder D was mounted into system <NUM>. However, it was found that under some conditions, CCD detector <NUM> saturated when sample holders A, B, or C were mounted into system <NUM>, thus rendering at least some data unusable. To solve this problem, a calibration procedure for adjusting operation of the LED <NUM> was developed. In certain embodiments, the calibration procedure comprises:.

The above calibration procedure was performed for sample holders A, B, and C of Table <NUM>, providing a duty cycle and/or drive current of LED <NUM> value for each. The calibration procedure accounted for random variations in power output and spectral characteristics existing between different individual LEDs <NUM> from the same manufacturer and sold under the same model or part number. The LED calibration procedure provided an image signal at CCD detector <NUM> that was approximately the same across various instruments <NUM> of the same design and construction, regardless of LED brightness and spectral characteristics for the particular LED <NUM> used in an individual instrument <NUM>.

Based on the calibration procedure, an LED a duty cycle or drive current value was stored in memory <NUM> of instrument <NUM> for each of the sample holder types A, B, and C shown in Table <NUM>. In general, it was found that the duty cycle or drive current value may be different for each of the sample holder types A, B, and C; however, in other embodiments, the duty cycle or drive current values may be the same for two or more sample holder types. It was also found that a duty cycle of <NUM>% could be used for sample holder D; however, in other embodiments, a duty cycle of less than <NUM>% may be stored from sample holder D. In addition, it was found that the duty cycle or drive currents stored for each of sample holders A, B, and C, and optionally D, for the instrument <NUM> could be used in all similar instruments configured the same, or essentially the same, as the instrument <NUM> of the exemplary embodiment. Alternatively, a calibration procedure may be performed on an individual instrument <NUM>, so that the duty cycle or drive currents stored for each of sample holders A, B, and C, and optionally D, are customized for that particular instrument.

As a result of the spectrum characteristics of LED <NUM>, it was discovered that an average sample fluorescence measurement time could be reduced by conducting the calibration procedure based on calibration data collected using channel <NUM> from Table <NUM>. For example, when the calibration procedure was conducted based on calibration data collected using channel <NUM>, it was discovered that more exposure time for other five filter channels was generally necessary during runs, leading to longer running time. When the calibration procedure is based on channel <NUM>, only <NUM> different exposure time are necessary for data collected on channels <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, and <NUM> exposure times is necessary only for channel <NUM>. More than one exposure time is used to increase the dynamic range of the data measurements made for a particular channel (excitation/emission wavelength band).

Regarding the use of more than one exposure time for channels <NUM>-<NUM>, when an end users runs, for example, a real time PCR, the sample volume can be different from user to user, and run to run; similar for sample concentration. Therefore, it is desirable to design instrument <NUM>' to provide a range of conditions for sample volume and sample concentration. If a single exposure time is provided, then for higher sample volumes and/or higher sample concentrations, detector <NUM> images may be saturated. Conversely, for lower sample volumes and/or lower sample concentrations, detector <NUM> images may be too low relative to noise levels. Thus, multiple exposure times may be used to extend the dynamic range of the system.

Regarding the use of channel <NUM> for calibration of LED <NUM>, it is possible to use any of the filter channels for this purpose. In the exemplary embodiment, the calibration target fluoresces with more strongly at wavelength within the band for channels <NUM> and <NUM>. Thus, channels <NUM> or <NUM> were preferred to perform calibration of LED <NUM>. In determining which of channels <NUM> and <NUM> to use for LED <NUM> calibration, Table <NUM> shows the average quant intensity (from detector <NUM> images) for sample holder B for different LED's used in different instruments of the same design and construction.

If channel <NUM> were used for LED <NUM> calibration, then all instruments would have about same image signal for channel <NUM>, but there a large variation in image signal produced when using excitation filters for each of channels <NUM> to <NUM>. For example, referring to Table <NUM>, using channel <NUM> for calibration, channel <NUM> would have a minimum Ch2/Ch1 ratio of <NUM> and a maximum Ch2/Ch1 ratio <NUM>. Thus, the ratio of the maximum Ch2/Ch1 ratio to the minimum Ch2/Ch1 ratio in Table <NUM> is <NUM>. The implication of this large variation is that three exposure times would be needed to provide the dynamic range covered using two exposure times for Ch1, based on calibration with Ch1. Similarly three exposure times would be needed for channel <NUM> to <NUM>. Thus, a total of 3x5+<NUM>=<NUM> exposure times are needed when all six channel (i.e., all six excitation/emission filter ranges) are used for measuring samples contained in sample holder <NUM>. The total time to provide emission data for all six channels is directly related to the total number of exposure times.

If we use channel <NUM> to perform the LED <NUM> calibration, the result is shown in Table <NUM>, based on the same set of instruments and LED's shown in Table <NUM>.

Calibrating LED <NUM> based on channels <NUM>-<NUM> all have about the same variation (bottom row of Table <NUM>) for the various LED's tested. Only channel <NUM> has a relatively large variation for the various LED's tested (ratio of max over min is <NUM> / <NUM> = <NUM>). Thus, <NUM> exposure times are needed for channel <NUM> to provide the same dynamic range as provided using two exposure times for channels <NUM>-<NUM>. As a result, a total of 2x5+<NUM>=<NUM> exposure times are needed, as compared to <NUM> exposure times when channel <NUM> is used to calibrate LED <NUM>. Thus, it has been discovered that the total time to provide emission data for all six channel may be reduced by using channel <NUM> to calibrate LED <NUM>, instead of channel <NUM>. Referring to the spectral function of LED <NUM> that is shown in <FIG>, it has been realized that the reason why channel <NUM> to <NUM> change with about same rate from LED to LED is that they fall in the broad spectrum peak. By contrast, channel <NUM> falls in a narrow peak, and shows different change rate from other channels from LED to LED. It has been discovered that this is a reason why calibration of LED <NUM> using channel <NUM> results in a lower number of image integration times to provide about the same dynamic range for six of channels <NUM>-<NUM>.

It was discovered that the thermal performance, power output, and spectral characteristics of LED <NUM> may vary in the instrument <NUM>, depending on which of sample holders A, B, C, or D was used. A variation in these parameters of between different instruments was also found due to random variations in LED characteristics between different individual LEDs <NUM> from the same manufacturer and sold under the same model or part number. In order to reduce the LED performance variation between sample holder types in a single instrument <NUM>, between different instruments <NUM> of the same design and construction, and/or between different LEDs in the same instrument, an LED thermal calibration procedure was developed.

Referring to <FIG>, various instruments <NUM> according to the exemplary embodiment were run under different conditions to determine a nominal target temperature, a low temperature limit, and a high temperature limit. Excitation source temperature control <NUM> comprised a fan that was configured to maintain LED <NUM> at a constant temperature or within a predetermined temperature range. Instrument <NUM> was configured to operate over a range of environmental conditions in which the ambient temperature in which the instrument was operated may be between <NUM> degrees Celsius and <NUM> degrees Celsius. It will be appreciated that embodiments of the present invention may be configured to operate within other temperature ranges, for example, between <NUM> degrees and <NUM> degrees Celsius, between <NUM> degrees and <NUM> degrees Celsius, between <NUM> degrees and <NUM> degrees Celsius, or the like. It will also be appreciated that in other embodiments, excitation source temperature control <NUM> may comprise other sources of controlling the LED temperature, for example, the use of a Peltier device or a liquid temperature controller.

In the current embodiment, instrument <NUM> was run by operating the LED using different conditions, including different sample holder types (sample holders A, B, C, or D from Table <NUM>), different instrument environment temperature (<NUM> degrees Celsius and <NUM> degrees Celsius), and different fan conditions (fan off (Fan DC = <NUM>) or <NUM> percent maximum fan drive voltage (Fan DC = <NUM>). The performance of different instruments <NUM> having different LEDs <NUM> is shown in <FIG>. <FIG> represents data obtained using sample holder A. <FIG> represents data obtained using sample holders B or C. <FIG> represents data obtained using sample holder D. Data points on the right side of each plot represent data obtain with instrument <NUM> in a <NUM> degree Celsius environment, while data points on the left side of each plot represent data obtain with instrument <NUM> in a <NUM> degree Celsius environment. The data points on the right side of the connecting lines is for the fan off condition, while data points on the left side of the connecting lines is for a fan drive voltage of <NUM> percent of the maximum.

Based on the data shown in <FIG>, suitable values for a nominal target temperature, a low temperature limit, and a high temperature limit for the different sample holders A, B, C, D are shown in Table <NUM>. These values were used to control the temperature of the LED for the various sample holders <NUM> used in a way that reduced variations in system performance and maintained a more consistent performance over a desired range of environmental operation conditions.

The instrument <NUM> is configured so that sample processing system <NUM> can receive, retain, or hold a first sample holder <NUM> (e.g., sample holders A, B, C, or D from Table <NUM>) comprising a first plurality of sample cells or units configured to hold a biological sample. Sample processing system <NUM> is also configured to receive, retain, or hold a second sample holder <NUM> (e.g., a different one of sample holders A, B, C, or D from Table <NUM>) comprising a second plurality of sample cells or units. For clarity, the current embodiment of system or instrument <NUM> will be referred to as system or instrument <NUM>', where it will be appreciated that the elements, features, and/or embodiments discussed above in relation to system or instrument <NUM>, where appropriate, may be incorporated into system or instrument <NUM>', or vice versa. System <NUM>' is configured to retain only one sample holder at a time or to retain a group of sample holders at a time that are all of the same type and construction. The first and second sample holders <NUM> are different from one another in at least one physical aspect. For example, a number, size, dimension, or volume of the sample cells for the first sample holder <NUM> may be different than that of the sample cells for sample holder <NUM>. Additionally or alternatively, the form or structure of sample cells for the first sample holder <NUM> may be different from that of sample cells for the second sample holder <NUM> (e.g., each may be made of a different material, or one of the sample holders may comprise through-holes that hold a liquid sample via capillary forces, while the other sample holder may comprise a microtiter plate comprising a plurality of wells or a microfluidics card comprising a plurality of sample chambers that are loaded via network of liquid flow channels). Sample processing system <NUM> may be configured to also retain one or more additional sample holders <NUM> (or sets of the same sample holder <NUM>) each comprising a plurality of sample cells, wherein the number of the sample cells in the additional sample holder or a characteristic dimension of the sample cells in the additional sample holder is different as compared to either the first sample holder <NUM> or the second sample holder <NUM>.

System <NUM>' further comprises excitation source <NUM> and excitation source temperature controller <NUM> including an excitation temperature sensor <NUM>, where excitation temperature sensor <NUM> is thermally coupled to the excitation source <NUM> so as to allow a temperature of the excitation source <NUM> to be measured or determined. System <NUM>' also includes an electronic processor <NUM> and a memory <NUM> and/or storage device <NUM> that includes data comprising a first target temperature for first sample holder <NUM> and a second target temperature for the second sample holder <NUM> that is different from or unequal to the value of the first target temperature. A memory or storage device <NUM>, <NUM> may also comprise instructions for execution by processor <NUM> to control a system temperature to the first target temperature when the first sample holder is retained by the instrument and to control a system temperature to the second target temperature when the first sample holder is retained by the instrument.

Excitation source <NUM> may be an LED, for example, as disclosed above in the exemplary embodiment.

A memory or storage device <NUM>, <NUM> may also comprise instructions for execution by processor <NUM> to determine if a target temperature (e.g., the first or second target temperature discussed above) of the excitation source can be maintained for a retained sample holder <NUM> (e.g., the first or second sample holders <NUM> discussed above). Referring to <FIG> a method <NUM> according an embodiment of the present invention includes a module <NUM> comprising selecting or determining a target temperature. Method <NUM> also includes a module <NUM> comprising operating excitation source <NUM> for a predetermined period of time, T. Method <NUM> also includes a module <NUM> comprising reading one or more temperatures from temperature sensor <NUM> during time T. Method <NUM> also includes a module <NUM> comprising determining if one or more of the sensed temperatures meet predetermined criteria. Method <NUM> also includes a module <NUM> comprising taking a first action if the predetermined criteria are not meet and a module <NUM> taking a second action if the predetermined criteria are meet. <FIG> is an exemplary flow chart <NUM>' of a specific embodiment of method <NUM>.

Claim 1:
An instrument (<NUM>) for biological analysis, comprising:
a sample processing system (<NUM>) configured to retain a first sample holder (<NUM>, <NUM>) comprising a first plurality of sample cells and a second sample holder (<NUM>, <NUM>) comprising a second plurality of sample cells, wherein at least one of (a) a number of the sample cells or (b) a characteristic dimension of the sample cells is different between the first sample holder and the second sample holder;
an excitation source (<NUM>) configured to produce one or more excitation beams;
an excitation optical system (<NUM>) configured to direct the one or more excitation beams (<NUM>) toward a plurality of samples retained by the sample processing system;
an optical sensor (<NUM>) configured to receive emission beams (<NUM>) from the first plurality of sample cells and configured to receive emission beams from the second plurality of sample cells;
an emission optical system (<NUM>) configured to direct the emission beams to the optical sensor;
an excitation source temperature controller (<NUM>) comprising a temperature sensor thermally coupled to the excitation source for determining the temperature of the excitation source,
an electronic processor (<NUM>); and
a storage device (<NUM>) including data comprising a first target temperature for the first sample holder and a second target temperature for the second sample holder that is unequal to the first target temperature, wherein the storage device comprises instructions to control the temperature of the excitation source to the first target temperature when the first sample holder is retained by the instrument and to control the temperature of the excitation source to the second target temperature when the second sample holder is retained by the instrument.