Lens system for collection of light for spectral analysis, and related devices, systems, and methods

Abstract

Optical systems and methods for collecting light use a collection lens system. An objective lens component of the collection lens system is configured to collect emitted light from a particle in an interrogation region, after irradiation with light from an illumination source. The objective lens component is configured to collect the light in differing spectral ranges and transmit the collected light in a direction of a z-axis of the lens system. An imaging lens component of the collection lens system is configured to receive the light from the objective lens component and transmit the received light in multiple paths corresponding to the differing spectral ranges to a plane at which a detector array is located. The collection lens system is configured to provide substantially constant magnification over a path of light transmitted through the collection lens system to the plane of the detector array.

Description

[0001] ATTORNEY DOCKET NO. TP387796WO1

[0002] LENS SYSTEM FOR COLLECTION OF LIGHT FOR SPECTRAL ANALYSIS, AND RELATED DEVICES, SYSTEMS, AND METHODS

[0003] TECHNICAL FIELD

[0004]

[0001] The present disclosure relates generally to lens systems for the collection of light from for use in spectral analysis. More specifically, the present disclosure relates to a collection lens system for collecting light from biological particles in flow cytometry instruments configured to perform spectral analysis. Related devices, systems, and methods also are disclosed.

[0005] INTRODUCTION

[0006]

[0002] Various biological sample analysis techniques involve analyzing the contents (e.g., cells, nucleic acids, or other particles in a biological sample) by directing light to the biological particles in the sample and measuring or otherwise detecting the behavior of the light scattered from the biological particles to analyze the characteristics of the biological particles. In flow cytometry, biological particles are suspended in a fluid and passed through a flow cell where they are exposed to an illumination source and detection of the light from the biological particles (e.g., scattered or, if absorbed and excited, emitted) then occurs at an interrogation region.

[0007]

[0003] In some applications, the biological particles of interest carry (are labeled with) fluorescent markers (also referred to as fluorophores) so that differing wavelengths of excitation light from the illumination source can be absorbed and then emitted in a band of wavelengths to thereby detect and identify the biological particles of interest. In the context of flow cytometry, such fluorescent labeling, excitation, and detection of the labeled biological particles can be used for spectral analysis (thus sometimes referred to as spectral flow cytometry). In spectral flow ATTORNEY DOCKET NO. TP387796WO1 cytometry, the emission spectra of each of the various fluorophores used to label biological particles are detected by a set of detectors (e.g., detector array) for subsequent analysis. The set of detectors can be, for example, a set of fiber optics, a set of charged-coupled diode imaging sensors, a set of complementary metal- oxide semiconductor CMOS) imaging sensors, or other photodetectors arranged in a defined spatial arrangement such as an array.

[0008]

[0004] To optimize the amount of light directed onto the detectors, relatively precise alignment is needed in x-y-z Cartesian space (with the z-axis generally defined along an optical axis) between the respective detectors and the respective light beams of different spectral or wavelength bands transmitted from a collection lens system that collects light emitted by the biological particles at the interrogation region of the flow cell. To achieve such alignment, as magnification of the light beams (spacing between adjacent light beams) transmitted to the detectors changes along the z-axis position, adjustment in pitch (or spacing between) detectors (e.g., fibers in a fiber array) may also need to be changed to avoid spot location errors (i.e., errors in which the light is not tightly focused / incident on the individual detectors and is offset from alignment). Such errors can result in not intercepting and collecting sufficient light at the individual detectors that is available in a light beam, which can lead to inaccuracies in downstream analysis.

[0009]

[0005] However, the pitch (spacing) used in various detector arrays (e.g., a fiber array) is generally fixed and such adjustment is not feasible, or at best complex, in a given system. Moreover, magnification is relatively sensitive to system setup and variations in the relative positioning between the interrogation region, collection lens ATTORNEY DOCKET NO. TP387796WO1 system, and detector array along the z-axis direction. For example, a relatively small variation of the location of the object (e.g., biological particle) in the object space of the collection lens system (e.g., position along the optical (z-) axis) relative to the collection lens system, can have a relatively large impact on the magnification of the light beams transmitted from the collection lens system at a given position along the z-axis. Such disparity in location of the object in the object space can occur, for example, by an alteration between the overall positioning of the flow cell relative to the collection lens system. In various systems, this positioning can be altered due to changes in an optical coupling element that is positioned between the flow cell and the lens system, among other things, as will be discussed further below.

[0010]

[0006] There is a continued need, therefore, to address the ability to provide a robust mechanism for alignment of the transmitted light beams from a collection lens system to the detector array to ensure sufficient light is intercepted at the detectors. Moreover, there is a continued need to reduce the amount of adjustment that may be needed in tuning the various optical components and detectors of a flow cytometry system so as to reduce the time needed to perform runs using such systems and to improve accuracy of the resulting measurements collected and analyzed.

[0011] SUMMARY OF ASPECTS OF DISCLOSURE

[0012]

[0007] Embodiments of the present disclosure may solve one or more of the above-mentioned problems and / or may demonstrate one or more of the above- mentioned desirable features. Other features and / or advantages may become apparent from the description that follows. ATTORNEY DOCKET NO. TP387796WO1

[0013]

[0008] Certain optical systems and methods for collecting light use a collection lens system. In one embodiment, an objective lens component of the collection lens system is configured to collect emitted light from a particle in an interrogation region, after irradiation with light from an illumination source. The objective lens component is configured to collect the light in differing spectral ranges and transmit the collected light in a direction of a z-axis of the lens system. An imaging lens component of the collection lens system is configured to receive the light from the objective lens component and transmit the received light in multiple paths corresponding to the differing spectral ranges to a plane at which a detector array is located. The collection lens system is configured to provide substantially constant magnification over a path of light transmitted through the collection lens system to the plane of the detector array.

[0014]

[0009] The optical system may comprise an optical coupling element optically coupling the interrogation region and the objective lens component. Moreover, modifying at least one dimension of the optical coupling element may cause a repositioning of a focal plane of the imaging lens component. The objective lens component may be configured to transmit separated light along multiple paths each having a ray angle divergence of seven degrees or less from the z-axis. The objective lens component may be configured to transmit the separated light along multiple paths each having the divergence angle of four degrees or less from the z- axis.

[0015]

[0010] Further, the objective lens component may be configured to transmit the separated light along the multiple paths having the divergence angle of seven ATTORNEY DOCKET No. TP387796WO1 degrees or less from the z-axis over a distance ranging from 25 - 300 mm. The imaging lens component may be spaced from the objective lens component along the z-axis at a distance greater than 25 mm.

[0016]

[0011] In one embodiment, the optical system includes an optical component arranged along the z-axis and positioned to receive light transmitted from the objective lens component. The one or more optical components may comprise one or more of a beam splitter, a dichroic mirror, or a filter.

[0017]

[0012] The imaging lens component may comprise a first imaging lens component and the system may further comprise a second imaging lens component. The optical component may be configured to receive and separate the light from the objective lens component into multiple beam directions, and to transmit the received light into the multiple beam directions angled respective to each other. The first imaging lens component and the second imaging lens component may be placed in respective paths of the multiple beam directions. A numerical aperture of the objective lens component may be equal to or greater than one. The collection lens system may be achromatic over wavelengths ranging from 350 nm to 1000 nm.

[0018]

[0013] In other embodiment, a spectral flow cytometer instrument comprises an interrogation region configured to contain a biological particles in a medium for spectral analysis, at least one illumination source arranged to transmit light toward the interrogation region to illuminate the particles, and a collection lens system.

[0019]

[0014] The collection lens system comprises an objective lens component configured to collect light from a biological particle in the interrogation region. An optical axis extends through the interrogation region and the objective lens ATTORNEY DOCKET NO. TP387796WO1 component and defines a z-axis of an x-y-z cartesian coordinate system. The objective lens component is configured to separate the collected light in differing spectral ranges and transmit the separated light in a direction of the z-axis of the lens system. The collection lens system further comprises an imaging lens component configured to receive the light from the objective lens component, and transmit the received light into multiple paths corresponding to the differing spectral ranges. The multiple paths are incident at respective predefined locations of a plane transverse to a direction of transmission of the multiple paths. The collection lens system may also comprise a detection array comprising a plurality of individual detectors arranged respectively at the predefined locations of the plane transverse to the direction of transmission of the multiple paths. The collection lens system may be configured to provide substantial telecentricity in an object space and an image space.

[0020]

[0015] The at least one illumination source may be at least one laser that provides a monochromatic light. Additionally, the at least one illumination source is at least one light emitting diode (LED) source arranged to transmit the light toward the interrogation region. The at least one illumination source may transmit light of varying wavelengths toward the interrogation region, wherein the wavelengths vary in a range from 350 nm to 1000 nm or in a range from 400 nm to 450 nm. The plurality of individual detectors may include at least one of a fiber optic array, charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS) sensor, and a red-green-blue (RGB) sensor. ATTORNEY DOCKET No. TP387796WO1

[0021]

[0016] In yet another embodiment, a method of collecting fluorescent and scattered light generated by a biological particle comprises collecting emitted light generated upon excitation of a biological particle, with light transmitted from an illumination source, at an objective lens component of a lens system. An optical axis extends through an interrogation region and the objective lens component and defines a z-axis of an x-y-z cartesian coordinate system. The method further comprises separating the light collected at the objective lens component into differing spectral ranges, transmitting, from the objective lens component, the separated light along a direction of the z-axis, receiving, by an imaging lens component of the lens system, the light transmitted from the objective lens component; and transmitting, from the imaging lens component, the received light along multiple paths corresponding to differing spectral ranges to a detection component. The multiple paths may be incident at respective predefined portions of the detection component, located in a plane transverse to a direction of transmission of the multiple paths. Additionally, magnification of the transmitted light may be maintained substantially constant from the objective lens component to detector component.

[0022]

[0017] The method may further comprise inserting an optical component in a free space between the objective lens component and the imaging lens component. The optical component in the free space may be a beam splitter that divides the light transmitted from the objective lens component along the direction of the z-axis into two light components. The detection component may be a first detection component, and the beam splitter may divide the light transmitted from the objective ATTORNEY DOCKET No. TP387796WO1 lens component in two separate paths, a first path leading to the first detection component and a second path leading to a second detection component. One of the two separate paths may be a scattered light component, and another one of the two separate paths may be a fluorescent light component.

[0023]

[0018] Additional objects, features, and / or advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present disclosure and / or claims. At least some of these objects and advantages may be realized and attained by the elements and combinations particularly pointed out in the appended claims.

[0024]

[0019] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims; rather the claims should be entitled to their full breadth of scope, including equivalents.

[0025] Brief Description of the Drawings

[0026]

[0020] The present disclosure can be understood from the following detailed description, either alone or together with the accompanying drawings. The drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate one or more exemplary embodiments of the present teachings and together with the description serve to explain certain principles and operation.

[0027]

[0021] FIG. 1 is a schematic view of an embodiment of a flow cytometry system in accordance with the present disclosure. ATTORNEY DOCKET No. TP387796WO1

[0028]

[0022] FIG. 2 is a schematic view of another embodiment of a flow cytometry system in accordance with the present disclosure.

[0029]

[0023] FIG. 3 is a schematic view illustrating optical coupling between a flow cell and a collection lens system.

[0030]

[0024] FIG. 4 is a schematic view of an embodiment of an arrangement of collection and detection optical components for use in a flow cytometry instrument in accordance with the present disclosure.

[0031]

[0025] FIG. 5 is a schematic view of another embodiment of an arrangement of collection and detection optical components for use in a flow cytometry system in accordance with the present disclosure.

[0032]

[0026] FIG. 6 is a schematic view of another embodiment of an arrangement of collection and detection optical components for use in a flow cytometry system in accordance with the present disclosure.

[0033]

[0027] FIG. 7 is a schematic view of an embodiment of an arrangement of a collection and detection optical components in a flow cytometry system for the purposes of illustration of various principles.

[0034]

[0028] Although the following detailed description makes reference to exemplary illustrative embodiments, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art and are contemplated as within the scope of the present disclosure and claims. Accordingly, it is intended that the claimed subject matter is provided its full breadth of scope, including encompassing equivalents. ATTORNEY DOCKET NO. TP387796WO1

[0035] Detailed Description of Various Embodiments

[0036]

[0029] This description and the accompanying drawings that illustrate various embodiments should not be taken as limiting. Various mechanical, compositional, structural, electrical, and operational changes may be made without departing from the scope of this description and the claims, including equivalents. In some instances, well-known structures and techniques have not been shown or described in detail so as not to obscure the disclosure. Like numbers in two or more figures represent the same or similar elements. Furthermore, elements and their associated features that are described in detail with reference to one embodiment may, whenever practical, be included in other embodiments in which they are not specifically shown or described. For example, if an element is described in detail with reference to one embodiment and is not described with reference to a second embodiment, the element may nevertheless be claimed as included in the second embodiment.

[0037]

[0030] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should ATTORNEY DOCKET No. TP387796WO1 at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0038]

[0031] It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0039]

[0032] This description’s terminology is not intended to limit the invention. For example, spatially relative terms — such as “beneath,” “below,” “lower,” “above,” “upper,” “proximal,” “distal,” and the like— may be used to describe one element’s or feature’s relationship to another element or feature as illustrated in the figures. These spatially relative terms are intended to encompass different positions (i.e., locations) and orientations (i.e., rotational placements) of a device in use or operation in addition to the position and orientation shown in the figures. For example, if a device in the figures is turned over, elements described as “below”, or “beneath” other elements or features would then be “above” or “over” the other elements or features. Thus, the exemplary term “below” can encompass both positions and orientations of above and below. A device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. In the orientation of the figures in the application, relative proximal and distal directions of the devices have been labeled. ATTORNEY DOCKET No. TP387796WO1

[0040]

[0033] Flow cytometry techniques, including those in accordance with various embodiments disclosed herein, can be used in immunology, oncology, hematology, microbiology, and stem cell research, among other biological analysis applications. Further, flow cytometry may be used for cell counting, cell sorting, immunophenotyping, cell cycle analysis, apoptosis detection, studying intracellular signaling pathways, among other applications. As biological particles, which can include but are not limited to, for example, cells, proteins, and / or nucleic acids, pass through an illumination source, they can scatter light in different directions or may absorb and emit electromagnetic energy (e.g., fluorescence) if labeled with fluorophores. This scattered and / or emitted light may then be collected and analyzed to characterize the particles based on various parameters.

[0041]

[0034] Proteins and other molecules (e.g. DNA) may be part of a cell or attached to a bead or particle to be detected and quantified by a flow cytometer. In that regard beads of various sizes may be analyzed. The beads can be labeled with fluorophores and can be used to bind to antibodies and other proteins or molecules (e.g. DNA). Further, in one embodiment, a flow cytometer can be used to count platelets.

[0042]

[0035] In addition to analyzing biological particles from biological samples, systems in accordance with the present disclosure can apply similar principles of operation to measure scattered and / or emitted light from a variety of other types of particles to perform similar counting, sorting, and / or characteristic analysis, and those having ordinary skill in the art would appreciate the disclosure is not limited to biological sample analysis applications. ATTORNEY DOCKET NO. TP387796WO1

[0043]

[0036] In various embodiment, the analyzed biological particles are various types of cells, such as blood cells, including white blood cells (leukocytes), red blood cells (erythrocytes), and platelets; immune cells, including T cells, B cells, natural killer (NK) cells, and dendritic cells; stem cells, including embryonic stem cells, hematopoietic stem cells, and mesenchymal stem cells; cancer cells, including tumor cells from solid tumors or leukemia / lymphoma cells; microbial cells, including bacteria, yeast, and other microorganisms, etc.

[0044]

[0037] In various embodiments, the flow cytometry system analyzes individual microbial cells or populations, indicative of microbial diversity, viability, metabolic activity, etc. Various species of bacteria may be analyzed based on size, shape, and fluorescent labeling. Certain species of yeast may be analyzed using the flow cytometry system for cell cycle analysis, viability assessment, and sorting. Moreover, the flow cytometry system may be used to study algal populations in environmental samples, aquatic ecosystems, and industrial processes.

[0045]

[0038] In yet other embodiments, the flow cytometry system is used for analyzing synthetic particles, such as microspheres, nanoparticles, and polymer particles.

[0046] Specifically, the system may analyze uniformly sized polymer microspheres or latex beads used as standards for calibration, quality control, and assay development. Further, the particles used for analysis may be engineered nanoparticles, including quantum dots, liposomes, and drug delivery vehicles. In addition, the flow cytometry system may analyze microparticles or nanoparticles made from polymers, used in drug delivery, diagnostics, and materials science. ATTORNEY DOCKET NO. TP387796WO1

[0047]

[0039] One embodiment involves using the flow cytometry system for evaluating subcellular structures or particles within cells, such as nuclei isolated from cells or tissues analyzed for DNA content, chromatin structure, and cell cycle progression. The examined particles may be fluorescently labeled organelles, such as mitochondria or endosomes, analyzed for abundance, size, and morphology.

[0048]

[0040] As discussed above, in spectral flow cytometry, collecting and guiding the light received from the biological particles onto the multiple detectors in a plane of a detector array can be challenging due to variance in light beam magnification along the optical (z) axis and the alignment needed with detectors. FIG. 7 provides an illustration of the challenges of alignment of multiple individual detectors of a detector array (such as a fiber array) and the spot (focus) errors that can occur.

[0049]

[0041] In FIG. 7, an interrogation region with an object from which light is emitted is shown at 7000. As an example, the object may be a biological particle emitting and / or scattering light as it passes through an interrogation region of a flow cell of a flow cytometry system. Upon impingement of the object with light from an illumination source (not shown), light in different wavelengths is scattered and / or emitted from the object 7000 and travels generally along a z-axis direction to be intercepted (received) by an objective lens component 7001 . In various configurations, the objective lens component 7001 is used to collect the light and gather it into multiple respective wavelength bands 7003 so that unique wavelength bands of light can be separated (e.g., in an x-y plane) as they are transmitted along the z-axis. Respective detectors, such as provided by a fiber optic array, for example, can then be positioned in a focus plane to intercept the separated ATTORNEY DOCKET NO. TP387796WO1 respective bands 7003 of light at a desired focus and measure the relevant signal therefrom, which can be analyzed for further analysis, as those having ordinary skill in the art are familiar with. In FIG. 7 the desired plane of focus is labeled as 7002b.

[0050]

[0042] But the collection lens system can result in relatively significant variation, depicted at 7006, in magnification (spacing in the x-y plane) of the separated light rays in the differing wavelength bands that depends upon the position along the z- axis. For example, if for the setup illustrated in FIG. 7, 7002b represents the desired plane of focus for the location of the 4 detectors of a detector array (shown by the solid circles in 7006), any relative displacement of the object 7000 and the objective lens component 7001 can consequently result in a displacement of the focus plane 7002b, for example, so that it causes the focus plane to be located at 7002a or 7002c relative to the light beams 7003 transmitted from the lens component 7001 . If the detectors of a detector array, however, are not adjusted in an x-y direction, then there is misalignment of where the light is focused relative to the detectors. Such misalignment (i.e., in the x-y direction) can cause spot errors 7005 as illustrated in FIG. 7, illustrated by the respective dotted lines above and below the solid to correspond to the positions of the change in the focus planes 7002a, 7002c. The respective separated light bands intended to be centered (tightly focused) on each individual detector (solid circles in the initial focus plane of 7002b) may instead be off the target in the x-y direction (shown by the dotted lines in the figures). These spot errors are a result of the magnification variation of the lens 7001 along the z-axis.

[0051] Even slight deviations can cause errors in measured signal. ATTORNEY DOCKET NO. TP387796WO1

[0052]

[0043] Variability in the flow cell, an optical coupling component (described further below with reference to FIG. 3), and / or the objective lens component manufacturing tolerances can result in magnification variations of over + / - 10%, and that in turn can cause spot errors that vary with distance of a respective wavelength band / detector pairing from the optical (z) axis, as reflected in the detailed view of FIG. 7 (comparing the focus positions of the light at the top and bottom in dashed in 7006 relative to the middle solid position for each band of light).

[0053]

[0044] Moreover, even with no magnification errors (due to manufacturing tolerances, etc.), it may still be necessary to align the detectors to the bands 7003 for maximum signal. This alignment for a system such as shown in FIG. 7 may be unfeasible by merely adjusting the position of the detectors in array alone in the direction of the z-axis, but the detector spacing, or pitch, may also need to be adjusted while adjustments along the z-axis take place.

[0054]

[0045] To address the issues discussed above and represented by the illustration of FIG. 7, aspects of the present disclosure therefore contemplate a collection lens system that can provide substantially constant magnification in image space, or along a distance spanning from the end of the lens system to the plane in which the detectors are, regardless of the location along the z-axis of the detectors. In other words, the spacing between adjacent light bands can be maintained without significant magnification variation as the position along the z-axis varies. This can allow for adjustment of the relative distance between the lens system and the plane of the detectors to achieve a desirable focus on, and thus more accurate detection of the separated light bands by, the respective individual detectors; in other words, ATTORNEY DOCKET No. TP387796WO1 focus of the separated light bands on individual detectors, without having to also adjust the pitch between the detectors or otherwise make corresponding adjustment of the detector locations in the x-y plane to avoid spot errors. This allows for the use of a detector array with a predefined x-y arrangement (e.g., pitch) between the individual detectors that is not dependent on a function of the location of the plane of the detector array relative to the lens system. Stated differently, various embodiments of collection lenses are substantially telecentric in object space and image space.

[0055]

[0046] Aspects of the present disclosure also contemplate providing a collection lens system in which the light beams in a free space defined by the lens system exhibit a relatively low range of divergence angles relative to the optical axis over a relatively long free space distance. This can permit the ability to place another optical element in the free space to allow for manipulating the light paths to travel at different directions while still allowing for each of the directions to provide substantially constant magnification (telecentricity) along a path to a location of a detector plane.

[0056]

[0047] These and other advantages and principles of operation will be understood further with reference to the figures and following description.

[0057]

[0048] FIG. 1 shows a schematic view of a flow cytometry system 100 which utilizes a lens system that provides substantially telecentric lenses. In the system 100, particles such as cells, microorganisms, nucleic acids, or synthetic particles carrying a biological component, etc., move from section A of a passage 103 to pass ATTORNEY DOCKET No. TP387796WO1 through a flow cell 101 , where they are exposed to illumination by an illumination source 102 prior to flowing through section B of the channel 103.

[0058]

[0049] In one embodiment, the illumination source 102 is one or more lasers that provide a monochromatic light that individually illuminates the particles 104 passing through the flow cell 101 . The choice of the illumination source 102 may depend on factors such as the wavelengths required to excite fluorophores that may be used to label the biological particle in the flow cell, the configuration of the system 100, the analytical capabilities of a detector, or a variety of other factors that one of ordinary skill in the art would be familiar with. The illumination source 102 may provide varying wavelengths.

[0059]

[0050] For example, the illumination source 102 may emit at various changeable wavelengths or can include multiple sources each emitting at differing wavelengths. For example, the illumination source 102 can comprise a plurality of lasers that each emit within a differing band of wavelengths, such as one or more of an ultraviolet (UV) wavelength ranging from about 350 nanometers (nm) to about 400 nm, a violet wavelength ranging from about 400 nm to about 450 nm, a blue wavelength ranging from about 450 nm to about 500 nm, a green wavelength ranging from about 500 nm to about 550 nm, a red wavelength ranging from about 600 nm to about 650 nm, or a laser of any other wavelength band deemed suitable for the application of interest. In another embodiment, the illumination source 102 is one or more light-emitting diodes (LED), which can emit light towards the flow cell 101 at specific wavelengths determined by the semiconductor materials and configuration used for the LED. The ATTORNEY DOCKET NO. TP387796WO1 selection of the LED can be made based on excitation wavelengths tailored to the specific application, such as fluorophores used and as discussed above.

[0060]

[0051] The system 100 further includes a collection lens system 105 comprising one or more lens components, including an objective lens component 105a and an imaging lens component 105b. Lens component as used herein is intended to include a series of lenses which together provide a specified manipulation of light through the lens component or a single lens that may include various other optical elements (e.g., including coatings, films, polishes, etc.) optically coupled with it to create the desired parameters described herein. The lens components of lens system 105 are configured to focus light onto a detector plane so as to register a relatively strong signal corresponding to the intensity of the light intercepted. The lens components of the lens system 105 may be of various focal lengths and types, including prime lenses, zoom lenses, and custom Lenses. In one embodiment, the lens components of the lens system 105 may include optical coatings to reduce glare, flare, and ghosting and enhance contrast and color fidelity and may provide fixed or variable focal lengths.

[0061]

[0052] Objective lens component 105a can be used for spectrally separating and magnifying (causing the respective wavelength bands of light beams to be spatially separated into plural distinct beams (paths)) the light from (scattered or emitted from) a particle 104 in the interrogation region of the flow cell 101 . In various embodiments, the objective lens component 105a can have a relatively high numerical aperture (NA) at the object and may correct for optical aberrations such as chromatic aberration, spherical aberration, etc. For example, the objective lens ATTORNEY DOCKET NO. TP387796WO1 component 105a can have an NA of greater than or equal to 1 at the object (e.g., the illuminated particle 104 in the flow cell 101 ).

[0062]

[0053] The objective lens component 105a may comprise multiple lens elements arranged in a specific configuration to achieve high-resolution imaging and minimize aberrations across a wide field of view, as will be discussed in further detail in regard to FIGs. 3-6 below. The objective lens component 105a may be configured to provide specific magnification levels and field of view characteristics which can be chosen depending on the parameters of the flow cytometry system 100, the object (e.g., biological particle) being analyzed, and a variety of other factors. In various embodiments, the objective lens component 105a is interchangeable with other objective lens component(s) exhibiting a different magnification level or other properties as may be selected, based on the teachings herein, to accommodate different requirements and imaging techniques of the system 100.

[0063]

[0054] In various embodiments, the objective lens component 105a is substantially telecentric. As used herein, a lens component that is substantially telecentric and variations thereof include a lens component in which the principal spatially separated rays of light that are transmitted through the lens component are substantially parallel to the optical axis extending from the particle and through the objective lens component 105a. In one embodiment, a principal ray or chief ray is the central ray of a collection of rays or light beam collected by the lens component 105a that passes through the center of the optical aperture stop of the system 100. In another embodiment, the optical aperture stop is placed at the back focal plane of the objective lens 105a for object space telecentricity. In yet another example, the ATTORNEY DOCKET No. TP387796WO1 aperture stop is the limiting aperture of the lens system 105 that limits the angular extent of the rays that can be captured, also known as the numerical aperture or NA.

[0064]

[0055] In this way, a substantially constant magnification can be achieved regardless of the distance of the flow cell 101 or object (particle 104) in the flow cell from the lens component 105a. This can provide minimal perspective distortion and enhance accuracy in ultimate measurement results (e.g., measurement of the intensity of the light at the detector). A lens component exhibiting substantial telecentricity can have its principal ray angles in a range of less than 2 degrees, less than 1 degree, less than 0.1 degrees, or less than 0.06 degrees relative to the optical (z-) axis, for example. The principal ray angles may vary with field and may be largest at the maximal field. In one embodiment, the lens system 105 has substantial object space telecentricity, as well as substantial telecentricity in the image space 107. Specifically, the principal rays from the last lens element to the detector plane may be parallel (telecentric) in the image space 107.

[0065]

[0056] In various embodiments, the distance between the flow cell 101 and the objective lens component 105a is defined by an optical coupling component 106 positioned between flow cell 101 and objective lens component 105a. In various embodiments, the optical coupling component 106 is an interchangeable component that can vary in thickness. For example, an optical coupling component 106 can be a gel slab that is somewhat compliant and thus can flatten as it is pressed for coupling between the flow cell 101 and the objective lens component 105a (as described further below with reference to FIG. 3). Replacing the optical coupling component 106 and / or modifying the amount a gel slab serving as the optical ATTORNEY DOCKET NO. TP387796WO1 coupling component 106 is pressed can thus vary the distance between the flow cell 101 and the objective lens component 105a, and in turn the distance between the object (particle in the flow cell 101 ) and the objective lens component 105a. Varying this distance can create a potential for a geometry of the optical elements and how they transmit / reflect light to be affected. Making the lens component 105a substantially telecentric can prevent the change in geometry from disrupting the magnification (spatial separation of the light beams) as a function of z-axis in the free space 122 between the objective lens 105a and the imaging lens 105b, as well as in the image space 107, which is a section of the flow cytometry system 100 between the imaging lens component 105b and a detector array 108. The telecentricity effect in the image space 107 is explained further below.

[0066]

[0057] In one embodiment, lens system 105 has substantial telecentricity in the image space 107 and substantial telecentricity also in the object space, between the zone of interrogation and the objective lens 105a. In the free space 122 between the objective lens 105a and imaging lens 105b, the rays (principal rays and marginal rays) may weakly diverge, allowing other optical components to be placed there with minimal impact to the optical performance. In another embodiment, the rays in the free space 122 are not collimated.

[0067]

[0058] As discussed above, the lens system 105 further includes the imaging lens component 105b, which is used to focus light onto a detector array 108, for example, but not limited to, a fiber array. In addition, the imaging lens component 105b can gather light rays from each of the spectrally and spatially separated bands transmitted from the objective lens component 105a and converge the rays in each ATTORNEY DOCKET NO. TP387796WO1 band to respective focused areas (e.g., detector locations in a plane of a detector array).

[0068]

[0059] The imaging lens component 105b, combined with the objective lens contributes to the performance of the lens system and can be manufactured in a variety of types, configurations, and focal lengths to accommodate different system and detector arrangements and conditions. The focal length of the image lens component 105b determines the lens system 105 Field of View (FOV) and magnification. In one embodiment, the imaging lens component 105b with a shorter focal length results in a smaller magnification and larger FOV, and in another embodiment, the imaging lens component 105b with a longer focal length results in a larger magnification and smaller FOV, for example.

[0069]

[0060] FIG. 2 shows another embodiment of a flow cytometry system 200 with a collection lens system in accordance with aspects of the present disclosure. Various elements of the flow cytometry system 200 are in common with those in the embodiment of FIG. 1 and thus are labeled with similar reference numerals except replacing the 100 series of numerals with a 200 series of numerals. Certain elements already described above will not be described here for the purposes of simplification. In the system 200, particles 204 being analyzed move from section A of a passage / channel 203 to pass through a flow cell 201 , where they are exposed to illumination by an illumination source 202 prior to flowing through section B of the channel 203.

[0070]

[0061] The illumination source 202 can be any of the illumination sources described and in an embodiment is a plurality of lasers that provide a ATTORNEY DOCKET NO. TP387796WO1 monochromatic light at differing wavelengths to illuminate the particles 204 passing through the flow cell 201 .

[0071]

[0062] The system 200 may include one or more detectors 214 for acquisition of data regarding the analyzed particles. In one embodiment, the detector 214 may be any of the detectors described above that can convert light into electrical signals so as to provide information useful for analysis of the particles 204.

[0072]

[0063] For example, the detector 214 can include a forward scatter camera (FSC) used for detecting scattering of light in a forward direction when a beam of light encounters particles or objects within the flow cell 201 . In one embodiment, signals measured by the FSC camera are used to analyze particles as they pass through the flow cell 201 . The forward scatter signal can provide information about the size and granularity of the particles. When a particle 204 in the channel 203 passes through a light beam emitted by the source 202, light is scattered in the forward direction, and the intensity of the forward scattered light can be measured by the FSC camera.

[0073]

[0064] The intensity of the forward scatter signal is indicative of the size of the particle, with larger particles scattering more light in the forward direction than smaller particles. For example, particles with more internal structures or granularity may scatter light differently compared to particles with smoother surfaces. Cells with internal structures and granularity can be detected using side scatter (SSC, 90 degrees to forward scatter or FSC direction) or large angle scattering. Different types of blood cells may form data clusters when viewed in FSC versus SSC plots. ATTORNEY DOCKET NO. TP387796WO1

[0074]

[0065] Other types of detectors can be used for 214, as well as various other detectors placed in different arrangements relative to the optical axis direction of transmission from an objective lens component 205a.

[0075]

[0066] The system 200 also includes a collection lens system 205 that includes the objective lens component 205a and an imaging lens component 205b, which can be similar to those described above with reference to FIG. 1 .

[0076]

[0067] In the embodiment of FIG. 2, however, the system 200 can include an optical component 209 can be inserted in the path after the objective lens component 205a to manipulate the beams of light from the objective lens in various ways. For example, to split beams of light exhibiting differing properties to be incident on two detector planes (e.g., comprising detectors 214 and 208). The telecentricity of the lens system 205, and properties of the objective lens component 205a-allow a relatively long free space to insert the optical component 209 without degrading the optical performance at the plane of the detectors such as a detector array 208, and detector 214, where light absorbed and emitted or scattered from particles 204 can be detected, as discussed further below.

[0077]

[0068] In various embodiments, the objective lens component 205a can include a relatively high numerical aperture (NA) and can correct for optical aberrations such as chromatic aberration, spherical aberration, etc. The objective lens component 205a may consist of multiple lens elements arranged in a specific configuration to achieve high-resolution imaging and minimize aberrations across a wide field of view, as will be discussed in further detail in regard to FIGs. 3-6. The objective lens component 205a may be configured to provide specific magnification levels and field ATTORNEY DOCKET NO. TP387796WO1 of view characteristics which can be chosen depending on the parameters of the cytometry system 200 or particular application / analysis of interest. The objective lens component 205a may be interchangeable to accommodate different magnification requirements and imaging techniques and analysis applications of the system 200.

[0078]

[0069] In one embodiment, the lens system 205 is substantially telecentric such that its magnification is substantially constant along the z-axis movement in image space, which includes along the local z’-axis from the reflection of the beams off the optical component 209 as shown in FIG. 2.

[0079]

[0070] In another embodiment, the principal rays of light are substantially parallel to the optical axis of the objective lens component 205a, i.e., the rays of light entering are substantially parallel to the lens component’s 205a optical axis. Accordingly, the cytometry system 200 can maintain substantially constant magnification regardless of flow cell 201 ’s or object 204’s distance from the lens component 205a, thus achieving minimal perspective distortion and accurate measurement results. In one embodiment, telecentricity is present in object and image spaces, but the rays in the free space may not be collimated or weakly divergent.

[0080]

[0071] As mentioned above, the distance between the flow cell 201 and the objective lens component 205a can be defined by an optical coupling component 206, such as a gel slab, used for optical coupling of the flow cell 201 with the objective lens component 205a. Replacing or modifying the optical coupling component 206 can vary the distance between the flow cell 201 and the objective ATTORNEY DOCKET No. TP387796WO1 lens component 205a, thereby creating a potential for a geometry of the optical elements to be affected. The telecentric properties of the lens system 205, can prevent such change in geometry from disrupting the magnification as a function of z-axis in image space 207, which is a section of the flow cytometry system 200 between the imaging lens component 205b and a detector array 208. The telecentricity effect on the image space 207 will be explained further below with reference to FIGs. 5 and 6.

[0081]

[0072] In the system 200, the imaging lens component 205b used to focus light onto a detector array 208, such as a fiber array, for example, can have an optical axis that is at an angle (e.g., orthogonal) to the principal optical (z) axis of the objective lens component 205a. For example, the optical axis from the optical element 209 to the imaging lens component 205b can be along the z’-axis and the plane of the detector array 208 can be in a plane that is parallel to the z-axis, as shown in FIG. 2. As with the imaging lens component 105b, the imaging lens component 205b can gather spectrally separated light rays and converge and focus the rays of each wavelength band to individual detectors of a detector array.

[0082]

[0073] The imaging lens component 205b may facilitate image sharpness and clarity, and may be manufactured in a variety of types, configurations, and focal lengths to accommodate different imaging arrangements and conditions. The focal length of the image lens 205b determines the lens system 205 Field of View (FOV) and magnification. In one embodiment, the lens 205b with a shorter focal length results in a smaller magnification and larger FOV, and in another embodiment, the ATTORNEY DOCKET NO. TP387796WO1 imaging lens component 205b with a longer focal length results in a larger magnification and smaller FOV for example.

[0083]

[0074] In an embodiment, the imaging lens component 205b is a microscope tube lens. The microscope tube lens can be used either with or without the optical component 209, such as a leaky mirror. In one embodiment, the tube lens transmits collimated input light, which requires using a tube lens that is greater in size. The imaging lens component 205b thus collects and distributes light reflected from the optical component 209 in order to ensure consistent illumination and advancement of light rays in separate wavelength bands to be focused on individual detectors of the detector array 208.

[0084]

[0075] In the system of FIG. 2, the optical component 209 is a “leaky mirror” which can selectively reflect or transmit light over a specific range of wavelengths. In one embodiment, the leaky mirror 209 allows a portion of incident light to pass through or “leak” while still reflecting another portion of the light. Specifically, the leaky mirror 209 may reflect light over different wavelengths toward the image lens 205b (e.g., tube lens), while allowing a portion of incident light to pass through (e.g., scatter) toward the detector 214.

[0085]

[0076] In various embodiments, the leaky mirror 209 includes a film or coating with controlled optical properties, such as thickness, refractive index, and layer structure. The leaky mirror 209 may be created based on these parameters in order to exhibit partial reflectance and partial transmittance at certain wavelengths, while maintaining high reflectivity at other wavelengths, for example. In one embodiment, the leaky mirror 209 is a dichroic mirror that reflects certain wavelengths while ATTORNEY DOCKET No. TP387796WO1 transmitting others. The dichroic mirror may separate excitation light from emitted fluorescence, allowing for simultaneous illumination and observation of the particles.

[0086]

[0077] Additionally, the flow cytometry system 200 may include one or more filters 211 a-c that filter incident light prior to letting a light component pass through. Certain examples of filters 21 1 a-c are blocking filters, bandpass filters 211 a, spatial filters 211 b, neutral density (ND) filters 211 c, etc. The bandpass filter 21 1 a may be an optical filter that selectively transmits light within a specific range of wavelengths, while blocking or attenuating light outside of that range. The spatial filter 211 b may selectively modify or enhance specific spatial frequencies within an image or optical signal by altering the distribution of light intensity across different spatial scales or frequencies. The ND filters 21 1 c may reduce the intensity of light without affecting its color or hue and may be gray or neutral in color. Further, as mentioned above, additional light path manipulation optical elements can be inserted to divert other aspects of the light beams transmitted from the optical element 209 to detector 214, with one such optical element 210 shown.

[0087]

[0078] The flow cytometry system 200 may further include one or filter flippers 212, such as, for example, polarizer filter flippers. The polarizer filter flipper 212 may be used to control the polarization of light passing through the system 200 by rotating or flipping a filter to adjust the orientation of polarization. The filter flippers 212 may be made of materials such as polarizing film or polarizing glass.

[0088]

[0079] Those of ordinary skill in the art would appreciate how various other optical elements (e.g., filters, mirrors, etc.) can be used within the main components ATTORNEY DOCKET NO. TP387796WO1 of the collection lens systems described herein to manipulate the light collected as desired without departing from the principles of operation discussed herein.

[0089]

[0080] Turning to FIG. 3, a schematic diagram of a flow cell 301 , an optical coupling element 306, and an objective lens component 305a that can be used in a flow cytometry system, such as the flow cytometry systems 100, 200, is illustrated and will be discussed for the purposes of illustrating various aspects of the present disclosure. The flow cell 301 includes a passage, such as a channel 303, in which a stream of particles can be flowed (such as in a liquid, gel, or other fluid), in a manner similar to that described above with respect to the embodiments of FIGs. 1 and 2.

[0090]

[0081] The flow cell 301 , which can be used as the flow cells 101 and 201 , can be made of any transparent material, such as, for example glass, plastic, etc. that allows light from the illumination source to reach the particles passing through channel 303. In some embodiments, the flow cell 301 is made with flat polished sides, and in other embodiments (not shown), the sides of the flow cell can be convex or concave or in any other shape considered appropriate for illumination by the source. The channel 303 may be a narrow, tubular pathway through which the cells or particles in the fluid stream flow. The channel 303 may be a flat sided channel, and in other examples, the shape of the channel 303 can be round, elliptical, or any other shape considered appropriate for illumination by the light source.

[0091]

[0082] In certain embodiments, hydrodynamic focusing is included in the channel 303 to facilitate flow of the cells or particles through the channel 303 in a monodisperse stream. The channel 303 provides an interrogation region for the ATTORNEY DOCKET NO. TP387796WO1 cells or particles to analyzed for properties such as forward scattering, side scattering, fluorescence emission, etc. The channel 303 provides an optical pathway for the interaction of the particles with the light emitted by the illumination source. Such a pathway can improve precision of the measurement of optical signals, such as scattering and fluorescence, emitted by the particles as they pass through the interrogation region.

[0092]

[0083] In an embodiment, the objective lens component 305a is a set of lenses 305n, which includes singlet or doublet spherical lenses (or triplet lenses or aspherical lenses in general) in a variety of combinations and arrangements. The lenses 305n may be placed in a housing 355 where they are aligned along their optical axes at predefined distances from each other and maintained at a fixed position respective to each other. The selection of the assembled lenses 305n may vary due to the materials, curvatures, and placements of the parts inside of the housing 355. In one example, the objective lens 305a includes four glass elements including two singlet lenses 305_1 and 305_2, and two doublet lenses 305_3 and 305_4, that are spherical in shape. However, this is a nonlimiting arrangement of lens types and numbers for an objective lens component in accordance with various embodiments of the present disclosure and other combinations are contemplated within the scope of the disclosure and can be selected to achieve the principles of operation disclosed herein.

[0093]

[0084] The objective lens component 305a may be used for transmitting light absorbed and emitted by or scattered from the particles included in the channel 303 and for transmitting spectrally separated and magnified wavelength bands of light to ATTORNEY DOCKET NO. TP387796WO1 be ultimately incident on a detector array. The objective lens component 305a may include a high NA and may correct for optical aberrations such as chromatic aberration, spherical aberration, etc. The individual lenses 305n may be arranged in a specific configuration to achieve high-resolution imaging and minimize aberrations across a wide field of view. The objective lens component 305a may be configured to provide specific magnification levels and field of view characteristics depending on the parameters of the flow cytometry system or other system in which it is deployed. The individual lenses 305n may be interchangeable to accommodate different magnification requirements and imaging techniques of the flow cytometry system.

[0094]

[0085] The objective lens component 305a is configured to be substantially telecentric, with the object space principal rays of light parallel to the optical axis of the objective lens component 305a, i.e., the rays of light entering the lens 305a may be substantially parallel to the lens component’s 305a optical axis. Accordingly, flow cytometry system, such as system 100 or 200, that incorporates the objective lens component 305a can maintain constant magnification along the z-axis regardless of the relative distance between flow cell 301 and the objective lens component 305a, thus achieving minimal perspective distortion and accurate measurement results.

[0095]

[0086] As discussed above with respect to the embodiments of FIGs. 1 and 2, FIG. 3 also includes an optical coupling component 306 in the form of a gel slab positioned between the flow cell 301 and the lens component 305a. The gap between the flow cell 301 and the objective lens component 305a caused by placement of the gel slab 306 can vary due to the soft, impressionable material of the gel that flattens when pressed for coupling between the flow cell 301 and the ATTORNEY DOCKET NO. TP387796WO1 objective lens 305a, and also due to manufacturing tolerances. The surface of the objective lens component 305a that faces the flow cell 301 contacts the gel slab 306 to provide the optical coupling between the two.

[0096]

[0087] In this manner, the gel slab 306 can be used to optimize the transfer of light from the flow cell 301 to the objective lens component 305a, by aligning the interrogation region of the channel 303 with the nearest one of the lenses 305n to maximize light transmission between them. However, due to replacement of the gel slab, as well as positioning and / or compression thereof, the gap between the flow cell and objective lens component 305a is variable, thereby creating a potential for the geometry of the optical elements to be affected.

[0097]

[0088] In addition to the variability of the gap between the flow cell 301 and the objective lens component 305a, the flow cell 301 , including the thickness of the fluid inside the cell 301 and / or the wall thickness of the cell 301 , also exhibits differing manufacturing tolerances. Such geometric variations may further vary the distance between the channel 303 that contains the particles and the objective lens component 305a. Similarly, the objective lens component 305a, and its individual lenses 305n, also exhibit variability in manufacturing tolerances. Consequently, the light that is collected by the objective lens component 305a may have a different magnification based on any combination of the varying tolerances.

[0098]

[0089] The configuration of the objective lens component 305a that provides for substantial telecentricity prevents such change in geometry (e.g., variation along the optical (z) axis) from disrupting the magnification as a function of position along the optical axis. In other words, the objective lens component 305a configuration that ATTORNEY DOCKET No. TP387796WO1 permits a substantially constant magnification along the optical (z) axis, allows for variability in manufacturing tolerances and / or system geometries (e.g., distances between the object space components).

[0099]

[0090] FIG. 4 shows another embodiment of a system 400 that includes a collection lens system and detector array, which, for example can be used in a flow cytometer system, such as the flow cytometer system 100. The collection lens system 405 includes objective lens component 405a and imaging lens component 405b. The objective lens component 405a may be a set of lenses 405n that further include singlet or doublet spherical lenses (or triplet or aspherical lenses in general) in a variety of combinations and arrangements. In one embodiment, there are four lenses 405n, two singlets and two doublets cemented together into a structure of six optical elements. In another embodiment, the imaging lens component 405b is a doublet comprising two optical elements, thereby amounting to eight optical elements total in the custom collection lenses 405 structure. The lenses 405 may be made by various glass grinding and polishing (or molding) methods that those of ordinary skill in the art are familiar with so as to achieve desired attributes.

[0100]

[0091] The arrangement of the optical lens elements can be modified and / or optimized by using ray tracing software. The software can include determination of the optimal lens forms and glass element choices or treatments for performance, such as collection efficiency, achromatic properties, magnification, and free space within the system 400 with minimal divergence angles. The ray tracing software can further optimize focusing conveyed to the fiber array 408 with the proper spot size and allowable collection angles for transmission to the fiber optic cables, for ATTORNEY DOCKET NO. TP387796WO1 example. The software can be modeled to allow input of parameters for the flow cytometry system 400 in order to reach the identified goals with a degree of tolerance. Some of the system parameters may be number, type, and shape of the lenses selected, the lens configuration, the free space between the lenses, the length and the width of the cytometer apparatus, the position and arrangement of the fibers, among other parameters. The ray tracing software is also used to ensure substantial telecentricity in image space and maintain substantial telecentricity in object space.

[0101]

[0092] In the embodiment shown in FIG. 4, the lenses 405n of the objective lens component 405a include two singlet lenses positioned on the object side of the objective lens component 405a (e.g., the side closest to the particle 404), with two additional doublet lenses placed on the imaging side of the objective lens component 405a (e.g., facing the imaging lens component 405b). The singlet lens 405n nearest to the object side may be semispherical. The flat side of the singlet lens 405n can face the object and can interface an optical coupling element, such as a gel slab, for example.

[0102]

[0093] The objective lens component 405a may be used for collecting light absorbed by and emitted or scattered from an object 404, such as, for example, a particle at an interrogation region of a flow cytometer system, and for further transmitting the light toward the imaging lens component 405b. The objective lens component 405a can have a high numerical aperture (NA) and correct for optical aberrations such as chromatic aberration, spherical aberration, etc. The lenses 405n are arranged in a specific configuration to achieve high-resolution imaging and ATTORNEY DOCKET No. TP387796WO1 minimize aberrations across a wide field of view. The objective lens component 405a also is configured to provide specified magnification levels and field of view characteristics, which can vary and be selected depending on the parameters of the flow cytometry system. The individual lenses 405n of the objective lens component 405a can be interchangeable to accommodate different magnification requirements and imaging techniques of the flow cytometry system.

[0103]

[0094] In accordance with aspects of the present disclosure, the lens system 405 may be optimized as a unit (objective lens + imaging lens) so that the lens system 405 is substantially telecentric while maintaining the desired magnification and a collection NA of approximately 1 .0, which allows the alignment of the fiber array 408 to the beams with independent z-axis and pitch adjustments. The lens system 405 may remain substantially telecentric in order to reduce the magnification changes due to the gel gap differences and other tolerances.

[0104]

[0095] With reference now to FIGs. 5 and 6, embodiments of arrangements of collection lens and detection components for use in spectral analysis of objects, such as in a flow cytometry system, in accordance with the present disclosure are schematically depicted. Various parameters and resulting effects of the light manipulation patterns associated with the arrangements of FIGs. 5 and 6 are discussed below. The embodiments of FIGs. 5 and 6 can be employed in a variety of spectral analysis applications, including flow cytometer systems, such as but not limited to the flow cytometer systems of FIGs. 1 and 2, respectively.

[0105]

[0096] FIG. 5 schematically illustrates an optical system 500 for use in spectral analysis applications which includes an arrangement of a collection lens and ATTORNEY DOCKET NO. TP387796WO1 detection components. The system 500 can be used for a variety of spectral analysis applications, including for example, in a flow cytometer system, such as, but not limited to the flow cytometer system of FIG. 1. The system 500 comprises a collection lens system 505, including objective lens component 505a and imaging lens component 505b, and a detector array 508. The various components are arranged so as to define an object space 521 , a free space 522, and an image space 507. Light reflected and / or emitted from an object 504 of interest, such as illuminated particles (for example at flowing through an interrogation region of a flow cell of a flow cytometer system), define the object space 521 from which the light is collected by the objective lens component 505a. The light can be any of the types of scattered or emitted light described above with reference to other embodiments. The objective lens component 505a collects the light and transmits the light toward a free space 522.

[0106]

[0097] In one embodiment, the light is telecentric in the object space 521 and the image space 507, where the principal rays are parallel to the z-axis minimizing magnification changes with movements along the z-axis and other tolerances. In another embodiment, the free space 522 has weakly diverging light, where the marginal ray angles are small such that the cone angles of the beams or NAs of the beams are small, while the principal rays have small angles relative to the z-axis. Such arrangement of the free space 522 is suitable for placing fold mirrors. In one embodiment, the maximum divergence from the optical axis in the free space 522 may range from 2 degrees to 8 degrees, or, for example, from 3 degrees to 5 ATTORNEY DOCKET NO. TP387796WO1 degrees. In the arrangement of FIG. 5, the light transmitted from the objective lens component 505a can reach the imaging lens component 505b in its totality.

[0107]

[0098] The objective lens component 505a can further be configured to transmit the light along multiple paths having a controlled range of divergence not to exceed a maximum value, noted above over a distance ranging from 25 mm to 300 mm. This distance represents the free space 522, or the distance over which the range of divergence of the transmitted light from the optical axis is maintained below or equal to a maximum range. In one embodiment, the distance of the free space 522 is greater than or equal to 25 mm and the divergence is less than 4 degrees over the free space 522. In another embodiment, the distance of the free space 522 ranges for 25 mm to 300 mm, and the maximum divergence ranges from 4 degrees to 7 degrees, for example, the free space may be approximately 85 mm with a maximum divergence of ranging from 4 degrees to 7 degrees over the free space 522.

[0108]

[0099] Providing a relatively long free space 522, enables flexibility in placement of the imaging lens component 505b along the along the optical axis (z-axis) so as to achieve desired focusing at the detector plane. For example, the imaging lens component 505b can be placed at a distance of greater than or equal to 25 mm from the end of the objective lens component 505a. For example, in an embodiment, the distance between the lenses 505a and 505b may be approximately 85 mm. In one embodiment, the imaging lens 505b is placed one focal length (of the imaging lens) from the system aperture to have image space telecentricity.

[0109]

[0100] The objective lens component 505a may collect the incoming light with relatively high collection efficiency. For example, the objective lens component 505a ATTORNEY DOCKET NO. TP387796WO1 can have a NA (numerical aperture) that is greater than or equal to 0.9, and in another example the NA can be greater than or equal to 1 . The numerical aperture of the objective lens component 505a defines the boundaries of a cone angle of light entering the collection lens system 500 and determine the system’s 500 ability to resolve fine details and capture light from the object 504. A higher numerical aperture (NA) corresponds to a wider cone of light entering the objective lens component 505a, thus enabling the objective lens component 505a to gather more light from the object 504.

[0110]

[0101] The objective lens component 505a is also achromatic over a relatively large range of wavelengths. For example, the objective lens component 505a is achromatic over wavelengths ranging from 350 nm in the ultraviolet wavelength band, across the visible spectrum, and to 1000 nm in the infrared wavelength band. The objective lens component 505a can bring two or more wavelengths of light to a common focus point and thereby minimize chromatic aberration by preventing different wavelengths (associated with different spectra or color) of light from focusing at different distances from the objective lens component 505a. In this manner, the objective lens component 505a can eliminate color fringing and improve the sharpness of the image acquired by the detectors 508 of the system 500.

[0111]

[0102] The objective and imaging lens components 505a and 505b of the system 500 can provide the ability to focus the spatially and spectrally separated light bands onto a detector array 508 with a desired spot size and desirable collection angles, which can be advantageous in particular with transmitting light down the individual fibers of a fiber detector array. Such a fiber array can be a linear (one-dimensional) ATTORNEY DOCKET No. TP387796WO1 array of multiple fibers, or a matrix (2- or more dimensional) array of fibers. A predefined pitch pattern can be a constant pitch or any other set spacing between individual detectors. In one embodiment, the pitch does not need to be adjusted regardless of the distance of a plane that includes the detector array to a plane of the coupling of the investigation zone to the lens system because the magnification of the lens system remains substantially constant.

[0112]

[0103] In one example the number of fibers is six fibers in a linear array, but the number may range from three to fifteen fibers. The adjacent arrays of fibers may utilize the same lens. The array 508 may include a core diameter of the fibers with a predetermined center-to-center spacing. The exemplary core diameter may be 600 microns, but the diameter may range from 200 microns to 1500 microns. Further, the center-to-center spacing among the fibers may range from 500-1500 microns, for example the center-to-center spacing can be about 800 microns.

[0113]

[0104] The telecentric arrangement of the lens components 505a and 505b results in the rays of light transmitted from the imaging lens component 505b remaining aligned in optimization to the plane of the detector array 508, or the optical fiber plane in the embodiment using the optical fiber array, as illustrated by FIG. 5. For the system 500, the magnification is maintained substantially constant as a function of position along the z-axis in the image space 507 extending to the detector array 508. As has been discussed, this permits the use of a predefined detector (fiber) pitch, which provides for robust focusing and reduction (or elimination) of spot location errors, regardless of changes in geometry (distance) between the various components along the optical axis. In one example, the ATTORNEY DOCKET No. TP387796WO1 alignment along the z-axis and the fiber array pitch are independent from each other, which facilitates the alignment.

[0114]

[0105] As shown in FIG. 5, the view of the x-y plane (labeled “P” in FIG. 5) represents three different z-positions of a plane in which a detector array can be positioned, i.e., illustrated by the dotted line, the solid line, and the dashed line. As illustrated by the view P, the arrangement of the collection lens system 505 allows the spatially separated light bands to be focused onto the individual detectors (shown by the solid circles in the view P), regardless of the position of the detector plane along the z-axis (i.e., as represented by the three different lines). Due to the telecentric arrangement of the collection lens system 505, the magnification remains substantially constant in the image space 507, hence, the spacing of the desired detection, so as to ensure accuracy of signal intensity captured, of the light rays does not change. Even in instances where the detector array spacing (spacing between detectors) also does not change, the detection is adequate and magnification variations do not occur along the z-axis. Being that the spacing of the arrays 508 matches the spacing of the light rays, the spot errors are substantially eliminated (or minimized) as a result, as shown in the “P” view of FIG. 5 (and in contrast with “P” view of FIG. 7).

[0115]

[0106] A collection lens system that has the properties relating to the free space discussed above with reference to FIG. 5 provides space to place an additional optical component in the free space due to the relatively long free space (near infinite conjugate section) that maintains the divergence of the rays to under a maximum range. For example, due to the properties associated with the free space, ATTORNEY DOCKET NO. TP387796WO1 optical components, such as a beam splitter, a dichroic mirror, a filter, etc. can be positioned in the free space and allow for additional light manipulation (and measurements) to occur.

[0116]

[0107] An embodiment of an arrangement of collection lens and detector components that incorporates an additional optical component in the free space is schematically illustrated in FIG. 6. As with the embodiment of FIG. 5, the optical system 600 includes an arrangement of collection lens, detection, and additional optical components, and can be used for a variety of spectral analysis applications, including for example, in a flow cytometer system, such as, but not limited to the flow cytometer system of FIG. 2.

[0117]

[0108] The system 600 comprises a collection lens system 605, including objective lens components 605a and imaging lens components 605b, and detector arrays 608. The various components are arranged so as to define an object space

[0118] 621 , a free space 622, and an image spaces 607. The system 600 includes an optical component 609 placed in a free space 622 after the objective lens component 605a, which has a configuration similar to the objective lens component 505a described above. The optical component 609 may be inserted in the free space 622 to create two principal beam directions P1 and P2 of light transmission (orthogonal to one another in the embodiment of FIG. 6, although other directions are considered within the scope of the present disclosure and may depend on the configuration of the optical component 609). Due to the properties of the free space

[0119] 622, which can be similar to those described above with reference to FIG. 5, the ATTORNEY DOCKET NO. TP387796WO1 optical element 609 is inserted along the z-axis of the system 600 where the light where rays are near collimated as has been described above.

[0120]

[0109] The optical component 609 may be a beam splitter or a dichroic mirror, or any other desired optical component placed in the free space 622. In one embodiment, the optical component 609 is a “leaky mirror” type of optical component that selectively reflects or transmits light in respective specific ranges of wavelengths. That is, the optical component 609 allows a portion of incident light to pass through or “leak” (for example in a first range of wavelengths) while reflecting another portion of the light (for example in a second range of wavelengths differing from the first range of wavelengths), such as a dichroic mirror, for example. In this way, the optical component 609 can transmit light in different wavelength ranges in separate beam directions P1 and P2 toward imaging lenses 605b_1 and 605b_2, respectively.

[0121]

[0110] The separated set of beams may travel through the imaging lenses 605b_1 and 605b_2, which allows for different final magnifications and path lengths for the two focusing arms created along directions P1 and P2. As noted above and shown in the schematic illustration of FIG. 6, the angle between the beam directions P1 and P2 can be 90 degrees but is not so limited and can be set at any other angle deemed suitable for packaging and folding the beams, for example to accommodate geometries of an overall system in which the system 600 may be employed, such as in a flow cytometry instrument.

[0122]

[0111] In various embodiments, the optical component 609 includes a film or coating with controlled optical properties, such as thickness, refractive index, and ATTORNEY DOCKET NO. TP387796WO1 layer structure. The optical component 609 may be created based on these parameters in order to exhibit partial reflectance and partial transmittance at certain respective wavelength ranges, while maintaining high reflectivity at other wavelength ranges, for example. In one embodiment, the optical component 609 is a dichroic mirror that reflects can separate excitation light from emitted fluorescence, allowing for simultaneous illumination and observation of the object 604 (e.g., particles in an interrogation region of a flow cell of a flow cytometer). In order to emit and detect fluorescence, a dichroic mirror may block the excitation or illumination light that is absorbed by the cells or the particles and allow the emitted fluorescence light to reach the detector. The emitted light may be at a longer wavelength than the excitation or illumination light, thus reducing background noise.

[0123]

[0112] The objective lens component 605a may be configured to transmit the received light to the imaging lenses 605b_1 and 605b_2 along multiple paths created by the optical element 609, each beam direction having a divergence in a range of less than 2 degrees to 8 degrees, for example, less than 3 degrees to 5 degrees, or for example, less than 4 degrees from the optical axis. In the embodiment of FIG. 6, the optical axis of beam direction P1 is y-axis of the system 600, and the optical axis of beam direction P2 is z-axis of the system 600.

[0124]

[0113] Moreover, the objective lens component 605a is configured to transmit the received light along one or more of the beam directions having a divergence over a free space that exhibits the same properties as discussed above with reference to the objective lens component 505a of FIG. 5. This can allow for the placement of the imaging lens components 605b_1 and 605b_2 relatively further from the objective ATTORNEY DOCKET No. TP387796WO1 lens component 605a and thus allow for various additional optical components to be placed between the objective lens component 605a and the respective imaging lens components 605b_1 and 605b_2. In one embodiment, the imaging lens components 605b_1 and 605b_1 are placed one focal length (of the imaging lens) from the system aperture to have image space telecentricity.

[0125]

[0114] The objective lens component 605a may have the same collection efficiency, numerical aperture, achromatic, and other properties as discussed above for the objective lens 505a. Further, the imaging lenses 605b_1 and 605b_2 may be similar to imaging lens 505b and the resultant focusing effects on the respective detectors 608_1 , 608_2 can therefore also correspond to that described for the array 508,

[0126]

[0115] In certain embodiments, the lenses 605a, 605b_1 , and 605b_2 of the system 600 may focus onto the arrays 608_1 and 608_2 of fiber optic cables with the proper spot size and allowable collection angles to transmit down the fiber arrays 608_1 and 608_2. In other embodiment, the termination points at the image planes of beam directions R1 and P2 are cameras, or a combination of fiber arrays and cameras, or any other detectors deemed appropriate, and broadly considered herein as detector arrays. The fiber arrays 608_1 and 608_2 may have the same arrangement, spacing, dimensions, core diameter, and other properties as discussed above for the array 508.

[0127]

[0116] As described above, in instances when an optical coupling element between the flow cell (not shown) and the objective lens component 605a is replaced or modified, the distance between the cell and the lens 605a may vary ATTORNEY DOCKET NO. TP387796WO1 along the Z-axis. However, the telecentric arrangement of the lens components 605a, 605b_1 and 605b_2 results in the rays of light transmitted from the imaging lens components 605b_1 and 605b_2 remaining aligned in optimization to the optical fiber plane. Consequently, the magnification is maintained constant as a function of the optical axes in the image spaces 607_1 and 607_2 extending to the fiber arrays 608_1 and 608_2. Therefore, a single fiber pitch can be used and determined independently from the x-y-z alignment, while practically eliminating any spot location errors.

[0128]

[0117] In one embodiment, the optical component 609 can be configured to separate the light into the beam directions P1 and P2 such that that fluorescent light emitted from the object 604 is conveyed along the reflected beam direction P1 and a forward scattered light is passed through and travels along the beam direction P2. The optical component can be any of the optical components 209 discussed above with reference to FIG. 2. The preceding is one exemplary embodiment however, and the optical element 609 can be configured to reflect and transmit any other wavelengths, polarization, or any combinations of wavelengths and polarization with controllable magnitude as desired.

[0129]

[0118] The system 600 may include one or more detectors for acquisition of data regarding the analyzed particles. In one embodiment, the detectors include a detector array 608_1 in the path of the imaging lens component 605b_1 that convert light into electrical signals, such as a fiber optic array, charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS) sensor, a red-green-blue ATTORNEY DOCKET NO. TP387796WO1

[0130] (RGB) sensor, etc. The image sensors may be used to acquire fluorescent signals reflected from the particles in the object 604.

[0131]

[0119] Moreover, the detectors may include a forward scatter camera (FSC), for example, for detector 608_2 in a path of the lens 605b_2 used for detecting scattering of light in a forward direction when a beam of light encounters the object 604. In one embodiment, signals measured by the FSC camera are used to analyze particles as they pass through the object 604. The forward scatter signal may provide information about the size of the particles. When a particle in the object 604 passes through a light beam emitted by the source, light is scattered in the forward direction, and the intensity of the forward scattered light may be measured by the FSC camera.

[0132]

[0120] The intensity of the forward scatter signal may be indicative of the size of the particle, with larger particles scattering more light in the forward direction than smaller particles. Additionally, the granularity or internal complexity of the particle may be ascertained from other large angle or side scatter (SSC) detectors. For example, particles with more internal structures or granularity may scatter light differently compared to particles with smoother surfaces.

[0133]

[0121] Embodiments of the disclosure can be used in a variety of applications and settings, including, as noted above in various flow cytometry systems. By way of nonlimiting example, various embodiments may be implemented in flow systems used for imaging flow cytometry or spectral flow cytometry.

[0134]

[0122] Further modifications and alternative embodiments will be apparent to those of ordinary skill in the art in view of the disclosure herein. For example, the ATTORNEY DOCKET No. TP387796WO1 systems and the methods may include additional components or steps that were omitted from the diagrams and description for clarity of operation. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the present teachings. It is to be understood that the various embodiments shown and described herein are to be taken as exemplary. Elements and materials, and arrangements of those elements and materials, may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the present teachings may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of the description herein. Changes may be made in the elements described herein without departing from the scope of the present disclosure and following claims.

[0135]

[0123] The nature of information, arrangements, and configurations depicted in the figures and described herein is exemplary. Those persons having skilled in the art would appreciate modifications to structure, dimensions, materials, and methodologies may be made without departing from the scope of the present disclosure.

[0136]

[0014] It is to be understood that the particular examples and embodiments set forth herein are nonlimiting, and modifications to structure, dimensions, materials, and methodologies may be made without departing from the scope of the present disclosure and claims including equivalents.

[0137]

[0125] Other embodiments in accordance with the present disclosure will be apparent to those skilled in the art from consideration of the specification and ATTORNEY DOCKET NO. TP387796WO1 practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with being entitled to their full breadth of scope, including equivalents.

Claims

ATTORNEY DOCKET NO. TP387796WO1WHAT IS CLAIMED IS:1 . An optical system comprising: a collection lens system comprising: an objective lens component configured to collect emitted light from a particle in an interrogation region, after irradiation with light from an illumination source, wherein an optical axis extends through the interrogation region and the objective lens component and defines a z-axis of an x-y-z cartesian coordinate system, and wherein the objective lens component is configured to collect the light in differing spectral ranges and transmit the collected light in a direction of the z-axis of the lens system; and an imaging lens component configured to: receive the light from the objective lens component, and transmit the received light in multiple paths corresponding to the differing spectral ranges to a plane at which a detector array is located, wherein the multiple paths are incident at respective predefined locations of a plane transverse to a direction of transmission of the multiple paths, and wherein the collection lens system is configured to provide substantially constant magnification over a path of light transmitted through the collection lens system to the plane of the detector array.ATTORNEY DOCKET NO. TP387796WO12. The optical system of claim 1 , further comprising: an optical coupling element optically coupling the interrogation region and the objective lens component, and wherein modifying at least one dimension of the optical coupling element causes a repositioning of a focal plane of the imaging lens component.

3. The optical system of claim 1 , wherein the objective lens component is configured to transmit separated light along multiple paths each having a ray angle divergence of seven degrees or less from the z-axis.

4. The optical system of claim 3, wherein the objective lens component is configured to transmit the separated light along multiple paths each having the divergence angle of four degrees or less from the z-axis.

5. The optical system of claim 3, wherein the objective lens component is configured to transmit the separated light along the multiple paths having the divergence angle of seven degrees or less from the z-axis over a distance ranging from 25 - 300 mm.

6. The optical system of claim 1 , wherein the imaging lens component is spaced from the objective lens component along the z-axis at a distance greater than 25 mm.ATTORNEY DOCKET NO. TP387796WO17. The optical system of claim 1 , further comprising: an optical component arranged along the z-axis and positioned to receive light transmitted from the objective lens component, wherein the one or more optical components comprise one or more of a beam splitter, a dichroic mirror, or a filter.

8. The optical system of claim 7, wherein the imaging lens component comprises a first imaging lens component and the system further comprises a second imaging lens component, wherein the optical component is configured to receive and separate the light from the objective lens component into multiple beam directions, and to transmit the received light into the multiple beam directions angled respective to each other, and wherein the first imaging lens component and the second imaging lens component are placed in respective paths of the multiple beam directions.

9. The optical system of claim 1 , wherein a numerical aperture of the objective lens component is equal to or greater than one.

10. The optical system of claim 1 , wherein the collection lens system is achromatic over wavelengths ranging from 350 nm to 1000 nm.ATTORNEY DOCKET No. TP387796WO11 1 . A spectral flow cytometer instrument, the instrument comprising: an interrogation region configured to contain a biological particles in a medium for spectral analysis; at least one illumination source arranged to transmit light toward the interrogation region to illuminate the particles; a collection lens system comprising: an objective lens component configured to collect light from a biological particle in the interrogation region, wherein an optical axis extends through the interrogation region and the objective lens component and defines a z-axis of an x-y-z cartesian coordinate system, an wherein the objective lens component is configured to separate the collected light in differing spectral ranges and transmit the separated light in a direction of the z- axis of the lens system; and an imaging lens component configured to receive the light from the objective lens component, and transmit the received light into multiple paths corresponding to the differing spectral ranges, wherein the multiple paths are incident at respective predefined locations of a plane transverse to a direction of transmission of the multiple paths; and a detection array comprising a plurality of individual detectors arranged respectively at the predefined locations of the plane transverse to the direction of transmission of the multiple paths, wherein the collection lens system is configured to provide substantial telecentricity in an object space and an image space.ATTORNEY DOCKET NO. TP387796WO112. The instrument of claim 1 1 , further comprising: a flow cell defining the interrogation region; and an optical coupling element between the flow cell and the objective lens component, wherein modifying at least one dimension of the optical coupling element causes a repositioning of a focal plane of the imaging lens component.

13. The instrument of claim 1 1 , wherein the objective lens component is configured to transmit the separated light along multiple paths each having a ray divergence angle of seven degrees or less from the z-axis.

14. The instrument of claim 13, wherein the objective lens component is configured to transmit the separated light along multiple paths each having the divergence angle of four degrees or less from the z-axis.

15. The instrument of claim 13, wherein the objective lens component is configured to transmit the separated light along the multiple paths having the divergence angle of seven degrees or less from the z-axis over a distance ranging from 25 - 300 mm.

16. The instrument of claim 1 1 , wherein the imaging lens component is spaced from the objective lens component along the z-axis at a distance greater than 25 mm.ATTORNEY DOCKET NO. TP387796WO117. The instrument of claim 1 1 , further comprising: an optical component arranged along the z-axis and positioned to receive light transmitted from the objective lens component.

18. The instrument of claim 17, wherein the optical component comprises one or more of a beam splitter, a dichroic mirror, or a filter.

19. The instrument of claim 17, wherein the imaging lens is a first imaging lens component, and the instrument further comprises a second imaging lens component, wherein the optical component is configured to receive and separate the light from the objective lens component into multiple beam directions, and to transmit the received light into the multiple beam directions angled respective to each other, and wherein the first imaging lens component and the second imaging lens component are placed in respective paths of the multiple beam directions.

20. The instrument of claim 1 1 , wherein a numerical aperture of the objective lens component is equal to or greater than one.21 . The instrument of claim 1 1 , wherein the at least one illumination source is at least one laser that provides a monochromatic light.ATTORNEY DOCKET NO. TP387796WO122. The instrument of claim 1 1 , wherein the at least one illumination source is at least one light emitting diode (LED) source arranged to transmit the light toward the interrogation region.

23. The instrument of claim 1 1 , wherein the at least one illumination source transmits light of varying wavelengths toward the interrogation region, wherein the wavelengths vary in a range from 350 nm to 1000 nm.

24. The instrument of claim 1 1 , wherein the at least one illumination source transmits light of varying wavelengths toward the interrogation region, wherein the wavelengths vary in a range from 400 nm to 450 nm.

25. The instrument of claim 1 1 , wherein the plurality of individual detectors includes at least one of a fiber optic array, charge-coupled device (CCD), a complementary metal- oxide-semiconductor (CMOS) sensor, and a red-green-blue (RGB) sensor.

26. A method of collecting fluorescent and scattered light generated by a biological particle, the method comprising: collecting emitted light generated upon excitation of a biological particle, with light transmitted from an illumination source, at an objective lens component of a lens system, wherein an optical axis extends through an interrogation region and the objective lens component and defines a z-axis of an x-y-z cartesian coordinate system;ATTORNEY DOCKET No. TP387796WO1 separating the light collected at the objective lens component into differing spectral ranges; transmitting, from the objective lens component, the separated light along a direction of the z-axis; receiving, by an imaging lens component of the lens system, the light transmitted from the objective lens component; and transmitting, from the imaging lens component, the received light along multiple paths corresponding to differing spectral ranges to a detection component, wherein the multiple paths are incident at respective predefined portions of the detection component, located in a plane transverse to a direction of transmission of the multiple paths, wherein magnification of the transmitted light is maintained substantially constant from the objective lens component to detector component.

27. The method of claim 26, the method further comprising: inserting an optical component in a free space between the objective lens component and the imaging lens component.

28. The method of claim 27, wherein the optical component in the free space is a beam splitter that divides the light transmitted from the objective lens component along the direction of the z-axis into two light components.ATTORNEY DOCKET NO. TP387796WO129. The method of claim 28, wherein the detection component is a first detection component, and wherein the beam splitter divides the light transmitted from the objective lens component in two separate paths, a first path leading to the first detection component and a second path leading to a second detection component.

30. The method of claim 29, wherein one of the two separate paths is a scattered light component, and another one of the two separate paths is a fluorescent light component.

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