Optical Train and Spectrum Edge Detection for an Imaging System

Abstract

The present disclosure relates generally to optical trains for imaging systems. More specifically, the present disclosure relates to imaging systems configured to limit optical aberrations. Furthermore, the present disclosure relates to methods of limiting optical aberrations in imaging systems configured to image a sample to detect individual detection components in a plurality of detection components.

Description

Technical Field

[0001] The present disclosure (the present invention) generally relates to a microscopic imaging system for the analysis of samples. In particular, the present disclosure relates to reducing optical aberrations of an imaging system for a fluorescence microscope. Further, the present disclosure relates to an optical train for an automated high-throughput imaging system. Additionally, the present disclosure relates to a method and an imaging system for sequentially detecting a number of detection components.

[0002]

Reference to Related Applications

[0003] All publications and patent applications mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Background Art

[0004] Microscopy is a powerful technique used to examine and image inorganic and organic samples. Examples of samples that may be selected include biological samples containing one or more biomarkers or components that may be targeted for detection or imaging. Current optical train type filters and imaging systems used in microscopy can use only a limited number of labels at any given time. Further, imaging systems have drawbacks such as image quality problems, for example, aberrations, spherical aberrations, focus shift, and image shift. As a result, medical technicians, researchers, and microscope users continue to seek systems and methods for imaging samples more efficiently and accurately.

Summary of the Invention

[0005] In one aspect, embodiments of an imaging system are provided. The imaging system includes at least one tiltable filter assembly including an optical filter configured to be positioned within the optical path of the imaging system, a dynamic correction optical system configured to be positioned within the optical path of the imaging system, and a fixed correction optical system configured to be positioned within the optical path of the imaging system.

[0006] The imaging system may further include a telecentric tube lens configured to be positioned within the optical path of the imaging system.

[0007] In some embodiments, the fixed correction optical system is configured to substantially reduce the spherical aberration caused by the combination of the optical filter and the dynamic correction optical system.

[0008] Even if residual spherical aberration occurs due to the combination of the fixed correction optical system, the dynamic correction optical system, and the optical filter, it can be said that such residual spherical aberration is substantially zero (0).

[0009] In some embodiments, with respect to a selected first incident angle of the optical filter, the dynamic correction optical system is configured to be tilted to a second incident angle, and the incident angle of the fixed correction optical system is configured to generate spherical aberration for substantially reducing the spherical aberration caused by the combination of the optical filter at the selected first incident angle and the dynamic correction optical system at the second incident angle. The dynamic correction optical system may be configured to substantially stabilize the lateral image shift caused by the tilting of the optical filter. In some embodiments, the sum of the lateral image shift caused by the dynamic correction optical system and the lateral image shift caused by the tilting of the optical filter is substantially constant. With respect to a selected first incident angle of the optical filter, the dynamic correction optical system may be configured to be tilted to a second incident angle, and the sum of the lateral image shift caused by the dynamic correction optical system and the lateral image shift caused by the optical filter is substantially constant.

[0010] In some embodiments, an optical filter held by at least one tiltable filter assembly and a dynamic correction optical system are configured to tilt about a substantially parallel X-axis, and a fixed correction optical system is tilted about a substantially perpendicular Y-axis. The telecentric tube lens may be disposed in the optical path of the imaging system at a position where the telecentric tube lens is telecentric in both the image and object spaces.

[0011] In some embodiments, the imaging system further includes a filter changer configured to hold at least one tiltable filter assembly. The filter changer may be a filter wheel, and the at least one tiltable filter assembly may be configured to tilt an optical filter to an incident angle selected from a range of approximately 0° to approximately 89.9°. In some embodiments, the filter changer includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 tiltable filter assemblies.

[0012] In some embodiments, the imaging system includes a low-incidence filter selected from at least one of the group consisting of a dichroic filter, a polychroic filter, a short-pass filter, a long-pass filter, a band-pass filter, a band-stop filter, and a multi-pass filter, and the low-incidence filter is configured to have an incident angle of excitation light selected from an angle in the range of approximately 10.0° to approximately 30.0°. The low-incidence filter may be a polychroic filter.

[0013] In some embodiments, the imaging system is a fluorescence microscopy imaging system.

[0014] The optical filter is preferably an interference filter.

[0015] In some embodiments, at least one tiltable filter assembly is configured to tilt the optical filter to an incident angle selected from a range of approximately 0° to approximately 89.9°.

[0016] In another aspect, a method for reducing the aberration of an imaging system is provided. The method includes positioning an optical filter in the optical path of the imaging system at a first incident angle, positioning a dynamic correction optical system in the optical path of the imaging system at a second incident angle, and positioning a fixed correction optical system in the optical path of the imaging system at a third incident angle, wherein the incident angle of the fixed correction optical system is configured to produce an aberration for substantially reducing the total aberration of the optical filter at the first incident angle and the aberration of the dynamic correction optical system at the second incident angle.

[0017] In yet another aspect, a method for stabilizing the lateral image shift of an imaging system is provided. The method includes positioning an optical filter in the optical path of the imaging system at a first incident angle and positioning a dynamic correction optical system in the optical path of the imaging system at a second incident angle, wherein the dynamic correction optical system is configured to substantially stabilize the lateral image shift caused by the tilt of the optical filter.

Brief Description of the Drawings

[0018]

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DETAILED DESCRIPTION OF THE INVENTION

[0019] In the following description, the term "original image" is used to represent an unprocessed image (whether or not it is to be visually displayed to an operator or end user) that includes data or at least one signal captured by a sensor or detector.

[0020] In the following description, the term "final image" is used to represent an image that includes processed data or at least one signal (whether it is to be visually displayed to an operator or end user or not). The "final image" may also be used to represent an image that is an output image obtained as a result of comparing and / or analyzing two or more original images or other final images (whether it is to be visually displayed to an operator or end user or not).

[0021] In the following description, the term "light" is not limited to the representation of electromagnetic lines in the visible portion of the electromagnetic spectrum, but is also intended to represent radiation in the ultraviolet and infrared portions of the electromagnetic spectrum.

[0022] In the following description, the term "sample" is used to represent an organic solid, an organic fluid, an inorganic solid, an inorganic fluid, a biological fluid, a biological semi-solid, a biological solid (which may remain as a solid, e.g., tissue, or may be liquefied in any suitable manner), a suspension, a portion of a suspension, a component of a suspension, etc.

[0023] In the following description, the term "target analyte" or "target substance" is used to represent a biological substance of interest.

[0024] In the following description, the term "non-target analyte" is used to represent a biological substance that is not a target analyte.

[0025] In the following description, the term "biomarker (biological indicator)" is used to represent a target analyte or a substance present on or within the target analyte or target substance (i.e., intracellular or extracellular target analyte internalized, for example, through phagocytosis within the target analyte). Biomarkers include, but are not limited to, peptides, proteins, subunits, domains, motifs, epitopes, isoforms, DNA, RNA, etc. A biomarker may preferably be a target molecule for dosing.

[0026] In the following description, the term "affinity molecule" is used to represent any molecule that can bind to or interact with another molecule. The interaction or binding may be covalent or non-covalent. Affinity molecules include, but are not limited to, antibodies, haptens, proteins, aptamers, oligonucleotides, polynucleotides, or any suitable molecule that interacts with or binds to another molecule (e.g., complementary molecules including, but not limited to, biomarkers, molecules of a binding pair, or biotin or avidin, etc.).

[0027] In the following description, the term "channel" is used to describe a color or color range based on signals provided by one or more detection components of a phosphor. The color or color range is obtained based on a selected interference filter (i.e., excitation filter, emission filter, polychroic filter, dichroic filter) and / or the wavelength of the signal. For example, the channel may be purple, blue, green, yellow, orange, red, engi color, etc. Further, when multiple channels are used, each channel has a unique color or color range. For example, the first channel may be green and the second channel may be orange. It should be noted that two or more detection components can provide signals exhibiting different wavelengths, but these signals should preferably be within the same channel based on the filter set used. For example, the first detection component provides a signal having a wavelength of 488 nm, and the second detection component provides a signal having a wavelength of 500 nm. Even if these wavelengths are not the same as each other, the filter set within one of the channels passes both the 488 nm and 500 nm wavelengths, thereby enabling imaging of both these wavelengths simultaneously, and thereby producing a single image including 488 nm emission and 500 nm emission. A channel can also describe a combination of an excitation bandpass (bandwidth) and an emission bandpass.

[0028] In the following description, the term "detection component" is used to represent a compound or substance that provides a signal for detection, thereby indicating the presence of another compound, another substance, an analyte, etc. in a sample or specimen. The detection component may be fluorescent, for example a fluorescent probe, or may be chromogenic, for example a chromogenic dye. The fluorescent probe may be a reactive dye, an organic dye, a fluorescent protein, a quantum dot, a non-protein organic molecule, a nanoparticle (e.g., nanodiamond), a phosphorus-integrated dot (PID), etc.

[0029] The detection component provides a signal for detection, thereby being a compound or substance that indicates the presence of another compound, another substance, an analyte, etc. in a sample or specimen. The detection component can be used as a tracer, as a label for a specific structure, as a label for a biomarker or otherwise. The detection component may be distributed, or the detection component can label an appropriate structure or biomarker in a manner including (but not limited to) ingestion, selective ingestion, diffusion, and attachment to binding molecules. The detection component may be bound to a biomarker by direct labeling or indirect labeling.

[0030] Examples of chromogenic dyes that can be used with various enzyme labels (e.g., horseradish peroxidase and alkaline phosphatase) include, but are not limited to, 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (CN), p-phenylenediamine dihydrochloride / pyrocatechol (Hanker-Yates reagent), Fast Red TR, new fuchsine, Fast Blue BB, etc. Examples of fluorescent probes include 1,5IAEDANS, 1,8-ANS, 4-methylumbelliferone, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthofluorescein, 5-carboxytetramethylrhodamine (5-TAMRA), 5-FAM (5-carboxyfluorescein), 5-HAT (hydroxytryptamine), 5-hydroxytryptamine (HAT), 5-ROX (carboxy-X-rhodamine), 5-TAMRA (5-carboxytetramethylrhodamine), 6-carboxyrhodamine 6G, 6-CR 6G, 6-JOE, 7-amino-4-methylcoumarin, 7-aminoactinomycin D (7-AAD), 7-hydroxy-4-methylcoumarin, 9-amino-6-chloro-2-methoxyacridine, ABQ, acid fuchsine, ACMA (9-amino-6-chloro-2-methoxyacridine), acridine orange, acridine red, acridine yellow, acriflavine, acriflavine foilgen SITSA, aequorin (luminescent protein), autofluorescent protein, Alexa Fluor350 (trademark), Alexa Fluor430 (trademark), Alexa Fluor488 (trademark), Alexa Fluor532 (trademark), Alexa Fluor546 (trademark), Alexa Fluor568 (trademark), Alexa Fluor594 (trademark), Alexa Fluor633 (trademark), Alexa Fluor647 (trademark), Alexa Fluor660 (trademark), AlexaFluor680™, Alizarin Complexone, Alizarin Red, Allophycocyanin (APC), AMC, AMCA-S, AMCA (Aminomethylcoumarin), AMCA-X, Aminoactinomycin D, Aminocoumarin, Aminomethylcoumarin (AMCA), Aniline Blue, Anthrosyl Stearate, APC (Allophycocyanin), APC-Cy7, APTRA-BTC, APTS, Astrazon Brilliant Red 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7GLL, Atabrine, ATTO-TAG™ CBQCA, ATTO-TAG™ FQ, Auramine, Aurphosphine G, Aurphosphine, BAO9 (Bisaminophenyl Oxadiazole), BCECF (high pH), BCECF (low pH), Berberine Sulfate, Beta Lactamase, BFP Blue-shifted GFP (Y66H: Blue Fluorescent Protein), BFP / GFP FRET, Bimane, Bisbenzamide, Bisbenzamide (Hoechst), bis-BTC, Blancophor FFG, Blancophor SV, BOBO™-1, BOBO™-3, Bodipy 492 / 515, Bodipy 493 / 503, Bodipy 500 / 510, Bodipy 505 / 515, Bodipy 530 / 550, Bodipy 542 / 563, Bodipy 558 / 568, Bodipy 564 / 570, Bodipy 576 / 589, Bodipy 581 / 591, Bodipy 630 / 650-X, Bodipy 650 / 665-X, Bodipy 665 / 676, Bodipy Fl, Bodipy Fl ATP, Bodipy Fl-Ceramide, Bodipy R6G SE, Bodipy TMR, Bodipy TMR-X Conjugate, Bodipy TMR-X,SE, Bodipy TR, Bodipy TR ATP, Bodipy TR-XSE, BO-PRO (trademark)-1, BO-PRO (trademark)-3, Brilliant Sulfoflavin FF, Brilliant Violet 421, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 786, BTC, BTC-5N, Calcein, Calcein Blue, Calcium Crimson (trademark), Calcium Green, Calcium Green-1, Calcium Green-2, Calcium Green-5N, Calcium Green-C18, Calcium Orange, Calcofluor White, Carboxy-X-Rhodamine (5-ROX), Cascade Blue (trademark), Cascade Yellow, Catecholamine, CCF2 (GeneBlazer), CFDA, CFP (Cyan Fluorescent Protein), CF405S, CF488A, CF488, CF543, CF647, CF750, CF760, CF780, FP / YFP FRET, Chlorophyll, Chromomycin A, Chromomycin A, CL-NERF, CMFDA, Coelenterazine, Coelenterazine cp, Coelenterazine f, Coelenterazine fcp, Coelenterazine h, Coelenterazine hcp, Coelenterazine ip, Coelenterazine n, Coelenterazine O, Coumarin pharoidin, C-Phycoerythrin, CPM Methylcoumarin, CTC, CTC Formazan, Cy2 (trademark), Cy3.18, Cy3.5 (trademark), Cy3 (trademark), Cy5.18, Cy5.5 (trademark), Cy5 (trademark), Cy7 (trademark), Cyan GFP, Cyclic AMP Fluorescent Sensor (FiCRhR), CyQuantCell proliferation assay, Dabsyl, Dansyl, Dansylamine, Dansylcadaverine, Dansyl chloride, Dansyl DHPE, DAPI, Dapoxyl, Dapoxyl 2, Dapoxyl 3, DCFDA, DCFH (dichlorodihydrofluorescein diacetate), DDAO, DHR (dihydrorhodamine 123), Di-4-ANEPPS, Di-8-ANEPPS, DiA (4-di-16-ASP), Dichlorodihydrofluorescein diacetate (DCFH), DiD - lipophilic tracer, DiD (DiIC18(5)), DIDS, Dihydrorhodamine 123 (DHR), DiI (DiIC18(3)), Dinitrophenol, DiO (DiOC18(3)), DiR, DiR (DiIC18(7)), DM-NERF (high pH), DNP, Dopamine, DsRed, DTAF, DY-630-NHS, DY-635-NHS, EBFP (enhanced blue fluorescent protein), ECFP (enhanced cyan fluorescent protein), EGFP (enhanced green fluorescent protein), ELF97, Eosin, ER-Tracker™ Green, ER-Tracker™ Red, ER-Tracker(Trademark) Blue-White DPX, Erythrosin, Erythrosin ITC, Ethidium Bromide, Ethidium Homodimer 1 (EthD-1), Ochracin, EukoLight, Europium(III) Chloride, EYFP (Enhanced Yellow Fluorescent Protein), Fast Blue, FDA, FIF (Formaldehyde-Induced Fluorescence), FITC, FITC Antibody, Flazo Orange: Fluo-3, Fluo-4, Fluorescein (FITC), Fluorescein Diacetate, Fluoroemerald, Fluorogold (Hydroxystilbamidine), Fluororuby, Fluoro-X, FM1-43 (Trademark), FM4-46, Flare Red (Trademark) (High pH), Flare Red (Trademark) / Fluo-3, Fluo-2, High Calcium, Fluo-2, Low Calcium, Fluo-2 / BCECF, Genacryl Brilliant Red B, Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, GeneBlazer (CCF2), GFP (S65T), GFP Red Shift (rsGFP), GFP Wild Type, Non-Ultraviolet Excitation (wtGFP), GFP Wild Type, Ultraviolet Excitation (wtGFP), GFPuv, Oxalic Acid, Granular Blue, Hematoporphyrin, Hoechst 33258, Hoechst 33342, Hoechst 34580, HPTS, Hydroxycoumarin, Hydroxystilbamidine (Fluorogold), Hydroxytryptamine, Indo-1, High Calcium, Indo-1, Low Calcium, Indodicarbocyanine (DiD), Indotricarbocyanine (DiR), Intracellular White CfJC-1, JO-JO-1, JO-PRO-1, LaserPro, Laurdan, LDS751, Leucohole PAF, Leucohole SF, Leucohole WS, Lissamine Rhodamine, Lissamine Rhodamine B, Calcein / Ethidium Homodimer, LOLO-1, LO-PRO-1, Lucifer Yellow, Lysotracker Blue, Lysotracker Blue-White Rhodotracker Green, Rhodotracker Red, Rhodotracker Yellow, Lysosensor Blue, Lysosensor Green, Lysosensor Yellow / Blue, Mag Green, Magdala Red (Floxine B), Mag-Fura Red, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1, Magnesium Green, Magnesium Orange, Malachite Green, Marina Blue, Maxilon Brilliant Flavin 10GFF, Maxilon Brilliant Flavin 8GFF, Merocyanine, Methoxycoumarin, Mitotracker Green, Mitotracker Orange, Mitotracker Red, Mitramycin, Monobromobimane, Monobromobimane (mBBr-GSH), Monochlorobimane, MPS (Methyl Green Pyronin Stilbene), mStrawberry, NBD, NBD Amine, Nile Red, Nitrobenzoxadiazole, Noreadrenaline, Nuclear Fast Red, Nuclear Yellow, Nile Blue Brilliant Thiavin E8G, Oregon Green (trademark), Oregon Green (trademark) 488, Oregon Green (trademark) 500, Oregon Green (trademark) 514, Pacific Blue, Pararosaniline (Fuchsin), PBFI, PE-Cy5, PE-Cy7, PerCP, PerCP-Cy5.5, PE-Texas Red (Red 613), Floxine B (Magdala Red), Horate AR, Horate BKL, Horate Rev, Horate RPA, Phosphine 3R, Photoresist, Phycoerythrin B, Phycoerythrin R, PKH26 (Sigma), PKH67, PMIA, Pontchrome Blue Black, POPO-1, POPO-3, PO-PRO-1, PO-PRO-3, Primuline, Procion Yellow, Propidium Iodide (PI), Pyrene, Pyronin, Pyronin B, Pyrozal Brilliant Flavin 7GF, QD400, QD425, QD450, QD500, QD520, QD525, QD530, QD535, QD540, QD545, QD560, QD565, QD570, QD580, QD585, QD590, QD600, QD605, QD610, QD620, QD625, QD630, QD650, QD655, QD705, QD800, QD1000, QSY7, Quinacrine Mustard, Red 613 (PE-Texas Red), Resorufin, RFP,RH414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B200, Rhodamine B extra, Rhodamine BB, Rhodamine BG, Rhodamine green, Rhodamine phalloidin, Rhodamine phalloidin, Rhodamine red, Rhodamine WT, Rose bengal, R-phycocyanin, R-phycoerythrin, rsGFP (red-shifted GFP (S65T)), S65A, S65C, S65L, S65T, Sapphire GFP, SBFI, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, Sevron Yellow L, sgGFP (trademark) (super glow GFP), SITS (primulin), SITS (stilbene isothiosulfonic acid), SNAFL calcein, SNAFL-1, SNAFL-2, SNARF calcein, SNARF1, Sodium green, Spectrum Aqua, Spectrum Green, Spectrum Orange, Spectrum Red, SPQ (6-methoxy-N-(3-sulfopropyl) quinolinium), Stilbene, Sulforhodamine B can C, Sulforhodamine G extra, SYTO11, SYTO12, SYTO13, SYTO14, SYTO15, SYTO16, SYTO17, SYTO18, SYTO20, SYTO21, SYTO22, SYTO23, SYTO24, SYTO25, SYTO40, SYTO41, SYTO42, SYTO43, SYTO44, SYTO45, SYTO59, SYTO60, SYTO61, SYTO62, SYTO63, SYTO64, SYTO80, SYTO81, SYTO82, SYTO83, SYTO84, SYTO85, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, Tetracycline, Tetramethylrhodamine (TRITC), Texas Red (trademark), Texas Red-X (trademark) conjugate, Thiadicarbocyanine (DiSC3), Thiazine Red R, Thiazole Orange, Thioflavin 5, Thioflavin S, Thioflavin TCN, Thiolite, Thiazole Orange, Tinopol CBS (Calcofluor White), TMR, TO-PRO-1, TO-PRO-3,TO-PRO-5, TOTO-1, TOTO-3, TriColor (PE-Cy5), Tetramethylrhodamine Isothiocyanate, TrueBlue, TrueRed, TubulinTracker (trademark) Green, UltraLight, Uranine B, UvitexSFC, wtGFP (wild type GFP), WW781, X-Rhodamine, XRITC, Xylene Orange, Y66F, Y66H, Y66W, Yellow GFP (yellow-shifted), Green Fluorescent Protein, YFP (Yellow Fluorescent Protein), YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3, CF350, CF405S, CF405M, CF405L, CF430, CF440, CF450, CF488A, CF503R, CF514, CF532, CF543, CF550R, CF555, CF568, CF570, CF583, CF594, CF620R, CF633, CF640R, CF647, CF660c, CF660R, CF680, CF680R, CF700, CF750, CF770, CF790, CF800, CF820, DY-344IN, DY-350XL, DY-360XL, DY-370XL, DY-376XL, DY-380XL, DY-395XL, DY-396XL, DY-480XL, DY-481XL, DY-485XL, DY-510XL, DY-511XL, DY-520XL, DY-521XL, DY-601XL, DY-350, DY-351, DY-405, DY-410, DY-415, DY-430, DY-431, DY-478, DY-488, DY-490, DY-495, DY-505, DY-530, DY-546, DY-547, DY-547P1, DY-548, DY-548P1, DY-549, DY-549P1, DY-550, DY-554, DY-555, DY-556, DY-557, DY-560, DY-580, DY-585, DY-590, DY-591, DY-594, DY-605, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, DY-641, DY-643, DY-647, DY-647P1, DY-648, DY-648P1, DY-649, DY-649P1, DY-650, DY-651, DY-652, DY-654, DY-660P1, DY-675DY-676, DY-677, DY-678, DY-679P1, DY-680, DY-681, DY-682, DY-684, DY-700, DY-701, DY-703, DY-704, DY-705, DY-706, DY-720, DY-730, DY-731, DY-732, DY-734, DY-736, DY-749, DY-747P1, DY-749P1, DY-750, DY-751, DY-752, DY-754, DY-765, DY-776, DY-777, DY-778, DY-780, DY-781, DY-782, DY-784, DY-800, DY-805, DY-820, DY-831, DY-845, DY-865 and combinations and derivatives thereof, including but not limited to. In one embodiment, the detection component, such as an organic phosphor, preferably has a molecular weight of about 0.01 kD or more, and such molecular weights include, but are not limited to, at least 1 kD, at least 10 kD, at least 25 kD, at least 50 kD, at least 75 kD, at least 100 kD, at least 150 kD, at least 200 kD, at least 250 kD, at least 300 kD, at least 340 kD, at least 350 kD, at least 500 kD, and at least 750 kD.,

[0031] In the following description, the terms "dye" or "label" are used interchangeably and are used to represent affinity molecules that bind to or interact with a detection component. The binding or interaction can be direct or indirect. Direct binding or interaction includes covalent or non-covalent interactions between a biomarker and a detection component. Indirect binding or interaction includes the use of at least a first and a second complementary molecule that form a binding pair. The first and second complementary molecules are a binding pair that binds or interacts in at least one of the following ways in combination: hydrophobic interaction, ionic interaction, hydrogen bonding interaction, non-covalent interaction, covalent interaction, affinity interaction, etc. Examples of binding pairs include immune-type binding pairs such as antigen-antibody reactions, antigen-antibody fragments, hapten-antihapten, or primary antibody-secondary antibody, non-immune-type binding pairs such as biotin-avidin, biotin-streptavidin, folic acid-folate binding protein, hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme-substrate analog, enzyme-pseudo substrate (substrate analog that cannot be catalyzed depending on enzyme activity), enzyme-cofactor, enzyme-modulator, enzyme-inhibitor, or vitamin B12-intrinsic factor, but are not limited thereto. Other suitable examples of binding pairs include complementary nucleic acid fragments (including complementary nucleotides, oligonucleotides, or polynucleotides), protein A antibodies, protein G antibodies, nucleic acid-nucleic acid binding proteins, polymer linkers (e.g., polyethylene glycol), or polynucleotide-polynucleotide binding proteins. The binding pair may be included in or used as an amplification method. The amplification method is also carried out to increase the number of detection components that bind to or interact with the biomarker so as to increase the signal. In one embodiment, when a binding pair is used, the dye is preferably pre-conjugated, e.g., already bound or interacting with the detection component when the affinity molecule is added to the sample during labeling, staining, or an additional step.In one embodiment, when binding pairs are used, the dyes may be conjugated within the sample, such that the labeling, staining, or additional steps include the introduction (in any desired or appropriate order) of the affinity molecule - first binding molecule conjugate, the second binding pair molecule - detection component conjugate, and the first and second binding pair molecules are complementary to and bind or interact with each other.

[0032] Furthermore, "multiple dyes" may be used to represent two or more dyes that are different from each other for the affinity molecule and / or detection component. For example, anti-CK-Alexa 647 is different from anti-EpCAM-Alexa 647. As another example, anti-CK-Alexa 647 is different from anti-CK-Alexa 488.

[0033] In the following description, the term "conjugate" is used to represent a first chemical substance, molecule, component, etc. that binds to or interacts with a second chemical substance, molecule, component, etc. The binding or interaction can be direct or indirect. Direct binding or interaction includes covalent or non-covalent interactions between a biomarker and a detection component. Indirect binding or interaction includes the use of at least a first and a second complementary molecule that form a binding pair. The first and second complementary molecules, in combination, are a binding pair that binds or interacts in at least one of the following ways: hydrophobic interaction, ionic interaction, hydrogen bonding interaction, non-covalent interaction, covalent interaction, affinity interaction, etc. Examples of binding pairs include immunological binding pairs such as antigen-antibody reactions, antigen-antibody fragments, hapten-antihapten, or primary antibody-secondary antibody, non-immunological binding pairs such as biotin-avidin, biotin-streptavidin, folic acid-folate binding protein, hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme-substrate analog, enzyme-pseudo substrate (substrate analog that cannot be catalyzed depending on enzyme activity), enzyme-cofactor, enzyme-modulator, enzyme-inhibitor, or vitamin B12-intrinsic factor, but are not limited thereto. Other suitable examples of binding pairs include complementary nucleic acid fragments (including complementary nucleotides, oligonucleotides, or polynucleotides), protein A antibody, protein G antibody, nucleic acid-nucleic acid binding protein, polymer linker (e.g., polyethylene glycol), or polynucleotide-polynucleotide binding protein.

[0034] In the following description, the term "signal" is used to represent an electric current or electromagnetic field that transmits data from one location or source to another location or detector. For example, a signal can be emitted by a detection component, thereby transmitting the presence of a sample or target analyte, such as a detection component on or inside a cell.

[0035] In the following description, the term "multiplexing" is used to represent a process or kit for labeling a sample with multiple dyes. Each of the detection components emits a different wavelength from the others. For example, at least two dyes can be used to label a sample. Multiplexing may include 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100 or more dyes.

[0036] An exemplary method of labeling a biomarker on a target analyte will be described. In one embodiment, a sample suspected of containing at least one target analyte is obtained. Suitable devices, systems, and / or methods for sample collection and / or processing include one or more of the following U.S. patents and U.S. patent application publications, namely, U.S. Patent Nos. 7,074,577; 7,220,593; 7,329,534; 7,358,095; 7,629,176; 7,915,029; 7,919,049; 8,012,742; 9,039,999; 9,217,697; 9,492,819; 9,513,291; 9,533,303; 9,539,570; 9,541,481; 9,625,360; 10,345,237; U.S. Patent Application Publication Nos. 2014 / 0161688; 2017 / 0014819; 2017 / 0059552; 2017 / 0074759; and U.S. Provisional Patent Application No. 62 / 873,390, each of which is incorporated herein by reference in its entirety. Devices, systems, and / or methods suitable for the recovery, isolation, or extraction of target analytes include one or more of the following U.S. patents and U.S. patent application publications, namely, U.S. Patent Nos. 9,222,953; 9,440,234; 9,519,002; 9,810,605; U.S. Patent Application Publication Nos. 2017 / 0219463; 2017 / 0276575, each of which is incorporated herein by reference in its entirety.

[0037] In one embodiment, the sample may be stained after sample collection and / or processing. In one embodiment, the sample may be multiplexed. At least one dye is added to the sample for labeling, for example, by an autostainer or manually by an operator. In one embodiment, at least one target analyte is stained. In one embodiment, at least one non-target analyte or non-target substance is stained. In one embodiment, at least one target analyte and at least one non-target analyte or substance are stained.

[0038] After staining, the sample may be imaged, thereby illuminating the stained sample with excitation light from a light source, such as a laser or light emitting diode, at one or more wavelengths, such as infrared, red, blue, green, and / or ultraviolet light. Imaging may be performed by a flow cytometer or microscope, such as a fluorescence microscope, scanner, or any other suitable imaging system or modality. In one embodiment, imaging is performed in a system that can provide signals across a spectrum including brightfield and / or darkfield illumination, fluorescence, etc. (not limited thereto) when the detection components are imaged. The generated images may be overlaid when multiple detection components are used. Emission, reflection, diffraction, scattering, and combinations thereof are used for detection / imaging. When analyzing the image, the target analyte can be detected, counted, and / or its location determined, for example, if it is desirable to recover or collect the target analyte. Imaging is performed in a tube, on a microscope slide, or in any suitable container or substrate for imaging.

[0039] The above method can be implemented by at least one of an imaging microscope, a scanner, a flow cytometer, or a microfluidic device, such as a chip or a microchannel, or the method can be implemented by any combination of the above. The method described can be used in a system that can provide signals over a spectrum including brightfield and / or darkfield illumination, fluorescence, etc. (not limited to these) when the detection components are imaged.

[0040] Optical train for imaging system In fluorescence microscopy, a phosphor (or fluorescent dye) is used to stain samples for examination or research, such as proteins or other molecules, tissues, and cells of interest. A phosphor can absorb light of one wavelength and emit light of another wavelength (fluorescence). In a typical fluorescence microscope setup, three filters are used: an excitation filter, an emission filter, and a dichroic filter. Each phosphor has a specific absorption or excitation wavelength band, and the excitation filter is selected to transmit that excitation wavelength range. Once excited, the phosphor emits light over a range of wavelengths. The emission filter transmits the desired emission wavelength. A dichroic filter, specially designed to reflect the excitation wavelength and transmit the emission wavelength, is used to separate the excitation channel from the emission channel. The dichroic filter may also be designed to reflect the emission wavelength and transmit the excitation wavelength.

[0041] FIG. 1 shows an embodiment of the optical train of a fluorescence microscope type imaging system. The optical path may include an excitation source 102 that emits light in at least one excitation light 104, such as in the visible spectrum, infrared (“IR”), or ultraviolet (“UV”) spectrum. Examples of the excitation source 102 include a laser light source, an LED light source, a xenon light source, a halogen light source, an incandescent light source, or other suitable light sources. In some embodiments, the excitation light 104 has a plurality of wavelengths including at least a first excitation wavelength 106 and a second excitation wavelength 108. In such embodiments, the excitation light 104 interacts with an excitation spectrum selector 110, and as a result, the first excitation wavelength 106 passes through the excitation spectrum selector 110, and the second excitation wavelength 108 is blocked from passing through the excitation spectrum selector 110. In certain embodiments, the excitation source selector 110 may include one or more optical filters or interference filters, such as one or more variable excitation filters. In such an embodiment, after the first excitation wavelength 106 exits the excitation filter of the excitation spectrum selector 110, this first excitation wavelength is then reflected by a second filter 112. The second filter 112 directs the direction of the first excitation wavelength 106 into the objective lens 114. The second filter 112 may be a dichroic, polychroic, short-pass, long-pass, band-pass, band-stop, or any suitable filter.

[0042] Continuing to refer to FIG. 1, the objective lens 114 receives the first excitation wavelength 106 reflected by the second filter 112 and focuses the first excitation wavelength 106 at a point or on the surface of the sample or a fraction 134 thereof, on, in, or near the sample or a fraction 134 thereof. The first excitation wavelength 106 stimulates a first detection component (not shown) on or in the sample or a fraction 134 thereof, whereby the first detection component (not shown) emits a first emission wavelength signal 116. The first emission wavelength signal 116 is captured by the objective lens 114, passes through the second filter 112 configured to pass the first emission wavelength, passes through the emission spectrum selector 130, and reaches the emission detector 140, where the original image is captured or collected. The emission detector 140 may be a charge-coupled device (“CCD”), a CMOS camera, a scientific CMOS camera, a photodiode, a photomultiplier tube, etc. for capturing image data, and the image data may then be compiled into an image and processed and analyzed by a computer or related software or program. In certain embodiments, the second filter 112 is a dichroic filter configured to reflect short wavelengths that typically excite a phosphor and transmit long wavelengths emitted by the phosphor. The second filter 112 and the emission spectrum selector 130 are configured together to limit or prevent non-emission energy and stray light from reaching the sensor of the emission detector 140.

[0043] In another embodiment, the excitation source 102 emits excitation light 104, which then interacts with the excitation spectrum selector 110. As a result, the second excitation wavelength 108 passes through the excitation spectrum selector 110, and the first excitation wavelength 106 is blocked from passing through the excitation spectrum selector 110. Next, the second excitation wavelength 108 is reflected by the second filter 112, thereby changing the direction of the second excitation wavelength 108 into the objective lens 114. The objective lens 114 receives the second excitation wavelength 108 and focuses the second excitation wavelength 108 onto a point or surface on, in, or near the sample or a fraction thereof 134. The second excitation wavelength 108 stimulates a second detection component (not shown) on or in the sample or a fraction thereof 134, thereby causing the second detection component (not shown) to emit a second emission wavelength signal 118. The second emission wavelength signal 118 is captured by the objective lens 114, passes through the second filter 112, passes through the emission spectrum selector 130, and preferably reaches the emission detector 140, where the original image may be captured. The described process may be performed one or more times for a desired number of detection components.

[0044] In a variant embodiment, the excitation source 102 may be configured to emit the excitation light 104 at one or more separate wavelengths, such as a first excitation wavelength 106 and a second excitation wavelength 108. In other embodiments, the optical path of the fluorescence microscope shown in FIG. 1 optionally does not include the excitation spectrum selector 110.

[0045] Continuing to refer to FIG. 1, the sample or a fraction thereof 134 may be disposed on the base 132 or between the cover 136 and the base 132. The cover 136 and the base 132 may be optically clear or optically transparent to enable imaging. In some embodiments, the base 132 and the cover 136 may be composed of one or more of glass, inert metals, metals, semimetals, organic or inorganic materials, and plastic materials, such as polymers, and combinations thereof.

[0046] Sample 134, cover 136, and base 132 may be disposed on platform 128 to move sample 134 in the x, y, or z direction as needed. Platform 128 may have an aperture 138 that can focus the first excitation wavelength 106 within, on, or near sample or a fraction thereof 134 by objective lens 114. Platform 128 may be driven by a drive device 120, which includes at least one of a z-direction drive device 124, an x-direction drive device 122, and a y-direction drive device 126 to position sample 134. Drive device 120 may be a motor, such as a servo motor or a stepping motor, a piezoelectric actuator, a solenoid, or the like.

[0047] The optical path may further include a cutoff aperture (not shown), such as provided in a confocal microscope, to increase the signal-to-noise ratio of the boundary optical signal.

[0048] In one embodiment, the excitation spectrum selector 110 or the emission spectrum selector 130 may be at least one fixed filter or at least one variable or tiltable filter configured to block or pass light of a desired wavelength. In one embodiment, the excitation spectrum selector 110 or the emission spectrum selector 130 may be a notch filter, a band-stop filter, a long-pass filter, a short-pass filter, a band-pass filter, or a polychroic filter. In one embodiment, the excitation spectrum selector 110 or the emission spectrum selector 130 may be a diffraction grating. In one embodiment, the excitation spectrum selector 110 or the emission spectrum selector 130 may include a variable angle or variable tilt filter that can be re-angled to block or pass the selected wavelength by changing the relative incident angle of the incoming excitation or emission light beam with respect to the filter. As an example, the first excitation wavelength 106 passes through the excitation spectrum selector 110, and the second excitation wavelength 108 is blocked from passing through the excitation spectrum selector 110, at least in part due to the angle of the excitation spectrum selector 110. As a variant, the excitation spectrum selector 110 may be selectively angled to block the first excitation wavelength 106 and pass the second excitation wavelength 108. As used herein, the incident angle is the angle (θ) between the light beam incident on the surface and the line perpendicular to the surface at the point of incidence.

[0049] In another embodiment, the first emission wavelength signal 116 passes through the emission spectrum selector 130, and the second emission wavelength signal 118 is prevented from passing through the emission spectrum selector 130, at least in part due to the angle of the emission spectrum selector 130. As a variant, the emission spectrum selector 130 may be selectively angled to block the first emission wavelength signal 116 and pass the second emission wavelength signal 118.

[0050] Continuing to refer to FIG. 1, the emission spectrum selector 130 may include at least one or more interference filters, or particularly emission filters, such as a first emission filter 142 and a second emission filter 144. The first emission filter 142 and the second emission filter 144 may be configured as variable filters that can be tilted or angled on an axis that is parallel to each other or perpendicular to each other as a variant. The first emission filter 142 may be tilted or angled to achieve a desired incident angle (shown by the filter 142 drawn with a long dashed line) between the first emission filter 142 and the emitted light ray. Similarly, the second emission filter 144 may be tilted or angled to achieve a desired incident angle (shown by the filter 144 drawn with a long dashed line) between the second emission filter 144 and the emitted light ray.

[0051] The first emission filter 142 and the second emission filter 144 can be tilted or angled independently of each other. In certain embodiments, the first emission filter 142 may be variably positioned to any desired position (i.e., the first position, the second position, the third position, the fourth position,... the nth position), and each position corresponds to a different angle θ. Similarly, the second emission filter 144 may be variably positioned to any desired position (i.e., the first position, the second position, the third position, the fourth position,... the nth position), and each position corresponds to a different angle θ. The first emission filter 142 and the second emission filter 144 may have independent positions and angles relative to each other, such that one or both of the first emission filter 142 and the second emission filter 144 can be angled or tilted at the same or different incident angles with respect to one or more emitted light rays. In a particular embodiment, the first emission filter 142 and the second emission filter 144 may be angled to pass or block emission of a desired wavelength.

[0052] For example, a first original image can be obtained with the first light-emitting filter 142 oriented at a first incident angle and the second light-emitting filter 144 oriented at a third incident angle. Next, the angle of the first light-emitting filter 142 can be redirected from the first incident angle to a second incident angle, during which the second light-emitting filter 144 remains in the state of the third incident angle. Then, a second original image can be obtained. In addition, when capturing at least one or more original images, the angle of the second light-emitting filter 144 can be redirected from the third incident angle to a fourth incident angle. Then, a third original image can be obtained. In one embodiment, at least two of the first, second, third, and fourth angles are the same. In one embodiment, none of the first, second, third, and fourth angles are the same.

[0053] Regarding any of the embodiments of tilting or angling at least one filter, tilting or angling any light-emitting filter for any desired time or sequence can block or pass a desired emission wavelength range. For example, after obtaining a first original image with the first light-emitting filter 142 oriented at a first incident angle, the first light-emitting filter 142 can be tilted or angled to a second incident angle. Then, a second original image can be obtained. Next, the first light-emitting filter 142 is tilted or angled again, and a third original image can be obtained. In other words, tilting or angling each filter at any point and by any amount independently of one or more other light-emitting filters can obtain any original image and / or any desired emission wavelength range.

[0054] In one embodiment, any number of filters can be used, and such numbers include, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100.

[0055] Some particular embodiments and implementations of the excitation spectrum selector 110 may include one or more variable interference filters configured to tune wavelength blocking and transmission by tilting the filter to change the angle of incidence of the optical path onto the filter. In some embodiments, at least one of the excitation spectrum selector 110 and / or the emission spectrum selector 130 may include at least one variable interference filter that can be tilted or angled. In certain such embodiments, both the excitation spectrum selector 110 and the emission spectrum selector 130 may include at least one variable interference filter. In such embodiments, the variable interference filter may be an excitation filter, an emission filter, a polychromic filter, and is configured to be used with a multi-channel fluorescence microscope and a high-throughput imaging system.

[0056] The individual filters of the excitation spectrum selector 110 or the emission spectrum selector 130 and / or the angle of incidence between these filters and the excitation or emission light beam may be selected to provide the desired wavelengths for capturing the original image within the selected bandwidth (bandpass) of the emission spectrum of one or more detection components. For example, the interference filters included in the excitation spectrum selector 110 and / or the emission spectrum selector 130 are configured to block and / or transmit the desired wavelengths along the optical path at locations on the lower and higher edges of one or more emission spectrum edges for capturing the original image. For example, in one embodiment, the detection components may have a spectral difference of 50 nm or less at these peaks. In one embodiment, the detection components may have a spectral difference of 10 nm or less at these peaks. In one embodiment, the detection components may have a spectral difference of 1 to 50 nm at these peaks. In one embodiment, the detection components may have spectra separated by only a few nanometers at these peaks, and such a few nanometers may include, but are not limited to, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 75 nm, 80 nm, 90 nm, or 100 nm. In one embodiment, the difference between successively positioned spectra (e.g., at the peaks) may be the same (e.g., the first detection component and the second detection component are separated by 10 nm, and the second detection component and the third detection component are separated by 10 nm). In one embodiment, the difference between successively positioned spectra (e.g., at the peaks) may be different from each other (e.g., the first detection component and the second detection component are separated by 10 nm, and the second detection component and the third detection component are separated by 25 nm).

[0057] In one embodiment, the incident angle θ of light to any filter may be approximately 0.0°, 1.0°, 2.0°, 3.0°, 4.0°, 5.0°, 6.0°, 7.0°, 8.0°, 9.0°, 10.0°, 11.0°, 12.0°, 15.0°, 20.0°, 25.0°, 30.0°, 40.0°, 45.0°, 50.0°, 60.0°, 70.0°, 75.0°, 80.0°, 85.0°, or 89.9°. In one embodiment, the incident angle of light to any filter may be up to 90° (but not including 90°). In one embodiment, the incident angle of light to any filter may be less than 90°. In one embodiment, the incident angle of light to any filter may be from 0.0° to 89.9°. In a particular embodiment, when two or more tiltable filters are provided, each filter can tilt freely and independently of each other, such that as a result, two or more filters can have the same incident angle or there are not two filters with the same incident angle. In a particular embodiment, the incident angle is selected according to the desired wavelength to be passed or blocked by the filter.

[0058] Filter changer The optical train of the imaging system disclosed herein may include a filter changer configured to quickly and easily exchange a number of optical filters. In certain embodiments, the filter changer may be a filter slider or filter wheel configured to hold one or more optical filters. The filter changer disclosed herein may be configured for use in systems and methods for multi-channel fluorescence microscopy and in automated high-throughput imaging systems. In some embodiments, at least one of the excitation spectrum selector 110 and / or the emission spectrum selector 130 may be configured to include at least one filter changer having one or more variable optical filters, such as one or more variable interference filters, that can be tilted or angled. For example, referring to FIG. 1, one or both of the first or second emission filters 142, 144 may be variable interference filters held by a filter changer having one or more variable interference filters.

[0059] Referring to FIG. 2A, one embodiment of the filter changer may be a rotating filter wheel. The filter wheel 200 may include one or more optical filters, such as a variable interference filter 242, which may be positioned within the optical path of the optical train. The filter wheel 200 may be configured to rotate clockwise or counterclockwise about or around an axis or axle at the hub 250. The filter wheel 200 may preferably include a position indicator or detent positioned at a desired position of the filter wheel 200. For example, the filter wheel 200 may be rotated manually or by a motor whose axle is attached to the hub 250, for the purpose of positioning the variable interference filter 242 at any desired filter position, such as filter position 252. In a preferred embodiment, the filter position 252 is positioned within the optical path of the light beam traveling through the optical train, whereby the variable interference filter 242 is positioned within the optical path of the light beam. For example, the filter wheel 200 may be rotated by a motor whose axle is attached to the hub 250 to position the variable interference filter 242 within the optical path of one or both of the first emission wavelength signal 116 and the second emission wavelength signal 118 (see also FIGS. 1 and 4B, for example).

[0060] In some embodiments, a filter changer, such as a filter slider or filter wheel 200, may have at least one filter assembly configured to hold an optical filter. As shown in FIG. 2A, the filter wheel 200 may have at least one filter assembly 260 configured to hold an optical filter, such as a variable interference filter 242. In certain embodiments, the filter wheel 200 can have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, and more filter assemblies 260. In one embodiment, the filter wheel 200 has two or more filter assemblies 260, and each filter assembly 260 includes an optical filter, and no two optical filters have the same optical filter characteristics. In another embodiment, each of the two or more filter assemblies 260 includes an optical filter, and at least two optical filters have the same optical filter characteristics. In yet another embodiment, the filter changer can have at least one filter assembly 260 without a filter, for example, the filter assembly 260 can consist of an optical window, clear glass, or the filter assembly 260 may hold nothing.

[0061] Referring to FIG. 2B, some embodiments of the filter assembly 260 are configured to hold an interference filter, such as the variable interference filter 242, within a case having a base 264 and a retainer 266. In one embodiment, the base 264 and the retainer 266 may include one or more hold-down clips 276 and provide a window 274 configured to allow light to pass through the variable interference filter 242. In certain embodiments, the base 264 may further have at least one bearing 278 configured to allow tilting, rotation, and / or translation of the filter assembly 260 while attached to the filter wheel 200. In one embodiment, the at least one bearing 278 is configured to allow tilting and / or translation of the filter assembly 260 including the variable interference filter 242, the base 264, and the retainer 266 while attached to the filter wheel 200 to change the angle of incidence between the variable interference filter 242 and the excitation or emission light beam in the optical path of the optical train.

[0062] Referring now to FIG. 3, in certain embodiments of an optical train for an imaging system including a filter wheel 200, a tilt mechanism may be configured to engage the filter assembly to tilt the filter assembly. For example, a tilt mechanism, such as a cam 300, may be configured to engage the filter assembly 260 while disposed in the optical path. In certain embodiments, the tilt mechanism, such as a cam 300, is configured to engage the bearing 272 of the filter assembly 260 and is configured to tilt the filter assembly 260 and the variable interference filter 242, thereby changing the angle of incidence between the variable interference filter 242 and the emission or excitation light beam in the optical path.

[0063] The cam 300 preferably has a body 302 and a tail portion 304 extending from the body 302, and the tail portion has an engagement surface 308 for engaging with the bearing 272 of the filter assembly 260. The tail portion 304 of the cam 300 can be of any suitable shape or form, for example, rectangular, cubic, triangular, pyramidal, curved, key-shaped, angled, or a combination thereof. Further, the tail portion 304 is preferably dimensioned and shaped to avoid any other components when the cam is rotated. In one embodiment of the cam 300, the body 302 has a bore 310 that extends at least partially through the body 302 for mating with the motor 320 or a connector to couple the motor 320 and the cam 300. In one embodiment, the body 302 has an axle or other connector for mating with the motor 320. In another embodiment, the cam 300 and the motor 320 may be an integral unit or a single component.

[0064] In one embodiment of the filter wheel 200, the filter assembly 260 and the variable interference filter 242 can be tilted at the filter position 252 by the engagement of the bearing 272 and the cam 300, and as a result, the incident angle of light on the filter can be any incident angle in the range from approximately 0.0° to approximately 89.9°. For example, the desired incident angle of light on the filter can be approximately 0.0°, 1.0°, 2.0°, 3.0°, 4.0°, 5.0°, 6.0°, 7.0°, 8.0°, 9.0°, 10.0°, 11.0°, 12.0°, 15.0°, 20.0°, 25.0°, 30.0°, 40.0°, 45.0°, 50.0°, 60.0°, 70.0°, 75.0°, 80.0°, 85.0°, or 89.9° (not limited thereto). In certain embodiments, when two or more variable interference filters are disposed in the optical path, each filter is preferably configured to tilt freely and independently of the other filters, and as a result, two or more filters can have the same incident angle, or there are not two filters having the same incident angle. The incident angle is preferably selected based on the desired wavelength to be blocked or passed by the filter.

[0065] Correction of optical aberration When light travels through the optical train of a fluorescence microscope as described herein, the light impinges on optical elements such as optical windows and optical filters made of glass or other substrates. Light rays incident on the glass surface of the filter will be distorted as they refract entering the glass from air and then return from the glass into the air. Refraction and distortion of light by the optical elements cause optical aberrations, and such optical aberrations can cause degradation of the performance of the optical system and image quality. Optical aberrations include spherical aberration, lateral image shift, focus shift, spherical aberration, chromatic aberration, coma (coma aberration), vignetting, and variable spectral selection across the field of view. Optical aberrations can cause image registration errors, focusing errors, and degradation of image resolution and accuracy. The occurrence of optical aberrations is particularly problematic when using multi-channel fluorescence microscope systems that utilize alignment of multiple images captured from various detection components, different phosphors, and filter sets. Embodiments of an optical train for a fluorescence microscope type imaging system are disclosed herein that minimize, limit, zero, and / or correct optical aberrations.

[0066] Referring to FIGS. 4A and 4B, an embodiment of an optical train for a fluorescence microscope system may include a tiltable or variable optical filter, such as variable interference filter 242, held by filter changer 400 and positioned in the optical path. In some embodiments, the variable optical filter may be used to select an emission band spectrum or emission wavelength because it generally provides significantly better transmission efficiency than other types of variable filters, such as those using liquid crystals. The excitation or emission light rays are refracted and distorted as they pass through the variable interference filter 242, thereby often causing lateral image shifts and spherical aberrations. Changing the angle of incidence of the variable interference filter 242 also changes any resulting lateral image shifts and spherical aberrations. As disclosed herein, optical elements may be disposed in the optical path to reduce, minimize, or correct the gradual optical aberrations caused by the variable interference filter 242. For example, in one particular embodiment, the dynamic correction optical system 410 may be configured to work with the variable interference filter 242 to produce predictable and substantially constant lateral image shifts and spherical aberrations for any desired angle of incidence of the variable interference filter 242. In other embodiments, the fixed correction optical system 420 may be positioned in the optical path and configured to substantially reduce or minimize any spherical aberrations caused by the variable interference filter 242 and the dynamic correction optical system 410. In other embodiments, the fixed correction optical system 420, the dynamic correction optical system 410, and the variable interference filter 242 may be positioned in the optical path and configured to simultaneously minimize spherical aberrations, lateral image shifts, and focus shifts.

[0067] Referring to FIG. 4B, an embodiment of the optical train may include a dynamic correction optical system 410 tiltable about an X-axis 412 that is substantially perpendicular to a Y-axis 422, and the fixed correction optical system 420 is tilted about the Y-axis 422. The dynamic correction optical system 410 may be a flat optically transparent plate, such as an optical window formed of a desired substrate. The dynamic correction optical system 410 can be positioned within the optical path of the optical train either before or after the variable interference filter 242.

[0068] In one embodiment, the dynamic correction optical system 410 is supported by a dynamic optical system assembly 414 that includes a hub 416. In certain embodiments, the hub 416 is disposed substantially along the X-axis 412. The dynamic correction optical system 410 can be selectively tilted about the X-axis 412 manually or by engagement of a motor and / or axle with the hub 416. In certain embodiments, the angle of incidence of light within the optical path relative to the surface of the dynamic correction optical system 410 can be any angle of incidence in the range from approximately 0.0° to approximately 89.9°. For example, the angle of incidence of light on the dynamic correction optical system 410 can be approximately 0.0°, 1.0°, 2.0°, 3.0°, 4.0°, 5.0°, 6.0°, 7.0°, 8.0°, 9.0°, 10.0°, 11.0°, 12.0°, 15.0°, 20.0°, 25.0°, 30.0°, 40.0°, 45.0°, 50.0°, 60.0°, 70.0°, 75.0°, 80.0°, 85.0°, or 89.9° (but not limited thereto).

[0069] Referring to FIGS. 4A and 4B, the fixed correction optical system 420 may be a flat optically transparent plate, such as an optical window formed on a desired substrate. The fixed correction optical system 420 is disposed in the optical path of the optical train at a fixed angle tilted about the Y-axis 422, and this angle ranges from approximately 0° to approximately 90°, from approximately 10° to approximately 80°, from approximately 20° to approximately 70°, from approximately 30° to approximately 60°, and from approximately 40° to approximately 50° (not limited thereto). In a particular embodiment, the fixed angle of the fixed correction optical system 420 is any angle that can substantially correct one or more of the optical aberrations that may be introduced by one or more optical elements in the optical train.

[0070] As described herein, the fixed correction optical system 420 is tilted about the Y-axis 422, and the dynamic correction optical system 410 is tilted about the X-axis 412. Referring to FIG. 4B, the variable interference filter 242 is tilted about an X-axis that is substantially parallel to the X-axis 412 and substantially perpendicular to the Y-axis 422. Thus, the variable interference filter 242 and the dynamic correction optical system 410 are configured to tilt about an X-axis that is substantially parallel and substantially perpendicular to the Y-axis 422, which is the center of the tilt of the fixed correction optical system 420.

[0071] The dynamic correction optical system 410 and the variable interference filter 242 can be tilted independently of each other. In certain embodiments, when the variable interference filter 242 is variably tilted to any desired position (i.e., various positions up to the first position, the second position, the third position, the fourth position, and the nth position), the emitted light of the desired wavelength can be passed or blocked, and each position corresponds to a different angle. Similarly, the dynamic correction optical system 410 can be variably tilted to any desired position (i.e., various positions up to the first position, the second position, the third position, the fourth position, and the nth position), and each position corresponds to a different angle. The dynamic correction optical system 410 and the variable interference filter 242 can have independent positions or angles relative to each other, such that one or both of the dynamic correction optical system 410 and the variable interference filter 242 can be tilted to the same incident angle or different incident angles with respect to one or more emitted light rays.

[0072] For example, during the capture of a number of original images, the first original image can be obtained with the variable interference filter 242 at a first incident angle and the dynamic correction optical system 410 at a corresponding third incident angle. For the second original image, the variable interference filter 242 can be re-angled from the first incident angle to the second incident angle, while the dynamic correction optical system 410 is tilted to the corresponding fourth incident angle. In this way, the dynamic correction optical system 410, working together with the variable interference filter 242, may be configured to produce a predictable and substantially constant lateral image shift and spherical aberration for any desired incident angle of the variable interference filter 242.

[0073] The variable interference filter 242, the dynamic correction optical system 410, and the fixed correction optical system 420 do not need to be positioned in the optical train in a particular order. In some embodiments, the optical path of the optical train passes through the fixed correction optical system 420, the dynamic correction optical system 410, and the filter wheel 200 in this order.

[0074] A. Spherical Aberration During operation of the fluorescence microscope, the optical train as described herein is configured to substantially reduce, minimize, and / or cancel out optical aberrations caused by one or more of the included optical elements. Continuing to refer to FIGS. 4A and 4B, in one embodiment, the fixed correction optical system 420 is configured to substantially reduce and / or make substantially zero the spherical aberrations introduced by both the dynamic correction optical system 410 and the variable interference filter 242. In another embodiment, the residual spherical aberration caused or introduced by the combination of the fixed correction optical system 420, the dynamic correction optical system 410, and the variable interference filter 242 is substantially zero (0).

[0075] In such an embodiment, the total spherical aberration caused by the dynamic correction optical system 410 and the variable interference filter 242 is defined to be approximately constant even when the variable interference filter 242 is selectively tilted about the X-axis. Thus, for a selected angle of the variable interference filter 242, the dynamic correction optical system 410 is also tilted such that these total spherical aberrations substantially cancel out the spherical aberration caused by the fixed correction optical system 420.

[0076] B. Lateral Image Shift In another embodiment, the sum of the lateral image shifts introduced by both the dynamic correction optical system 410 and the variable interference filter 242 is maintained substantially constant for any desired angle of incidence of the variable interference filter 242. For example, the total lateral shift caused by the dynamic correction optical system 410 (LSHFT 410 ) and the variable interference filter 242 (LSHFT 242 ) is substantially constant (LSHFT Constant ). In another form, it is as follows. LSHFT Constant =LSHFT 410 +LSHFT 242

[0077] Thus, for a selected angle of incidence of the variable interference filter 242, the dynamic correction optical system 410 (LSHFT 410) and the total lateral image shift caused by the variable interference filter 242 (LSHFT 242 ) is approximately a constant value even if the variable interference filter 242 is selectively tilted about the X-axis.

[0078] C. Focus Shift In another embodiment, the optical train as described herein is configured to substantially stabilize the total focus shift caused by the refractive path focal length provided by the dynamic correction optical system 410 and the variable interference filter 242. When the focus shift is stabilized, the system focus may be offset by adjusting the sensor or camera and / or the objective lens along the optical axis to achieve the desired image focus.

[0079] Telecentric tube lens An infinity-corrected objective lens is a microscope objective lens that has a focus at infinity. To form an image with an infinity-corrected objective lens, it is advisable to use a tube lens to focus the image on the image plane. One advantage of using an infinity-corrected objective lens together with a tube lens is that there is space to insert additional optical components, such as optical filters, between the objective lens and the tube lens into the system. In some embodiments described herein, a telecentric tube lens configured to be used with an infinity-corrected objective lens should be positioned within the optical path of the fluorescence microscope to minimize, reduce, limit, zero, and / or correct optical aberrations such as vignetting, chromatic aberration, spherical aberration, and variable spectral selection across the field of view. Also, using a telecentric tube lens can minimize the need for digital correction of optical aberrations, thereby enabling the image processing to operate quickly by reducing the CPU load, resulting in a high system throughput.

[0080] Referring to FIG. 5, an embodiment of an optical train for a fluorescence microscope system may include a double telecentric tube lens 500. A telecentric tube lens is a compound lens having an entrance pupil or an exit pupil at infinity. The telecentric tube lens produces chief rays that are parallel to the optical axis of the optical path. Chief rays that are parallel to each other are desirable because when they pass through a tiltable or variable optical filter, such as variable interference filter 242 (FIG. 4B), they produce a constant selected spectrum across the entire field of view. In other words, the parallel chief rays created by the telecentric tube lens have the same angle of incidence with respect to the variable optical filter and thus generally exhibit the same spectral response with respect to the variable optical filter.

[0081] In certain embodiments, the double telecentric tube lens 500 is configured to be disposed at an upstream position 510 as viewed from a tiltable filter or variable interference filter, such as variable interference filter 242. The position 510 may be disposed within the optical path, at which position the double telecentric tube lens 500 is telecentric in both the image space and the object space. In such embodiments, the double telecentric tube lens 500 produces chief rays that are substantially parallel through the image space (the space between the lens and the detector). In similar such embodiments, the double telecentric tube lens 500 is configured to produce chief rays that are substantially parallel and have an angle of incidence of approximately zero (0) at the emission detector 140. Since the chief rays are parallel to each other, variable spectral selection, spherical aberration, chromatic aberration, coma (coma aberration), and vignetting across the entire field of view can be minimized or limited, thereby improving image resolution and quality.

[0082] Low incidence filter Generally, dichroic filters and polychroic filters are configured to have a desired passband and stopband designed to operate at a standard incident angle of 45°. However, when the incident angle is small, good filter performance can be obtained, and such good filter performance includes blocking, transmission, sharp bandpass (band-pass) edges, and improved beam collimation. This specification discloses embodiments of an optical train for a fluorescence microscope that includes a polychroic excitation filter having an incident angle of excitation light that is smaller or narrower than the standard 45° incident angle.

[0083] Referring to FIG. 6, the optical path preferably includes an excitation source 102 that emits excitation light 104 directed toward an excitation spectrum selector 110, and the excitation spectrum selector 110 includes at least one excitation filter configured to transmit a desired excitation wavelength, such as a first excitation wavelength 106 and / or a second excitation wavelength 108. After the first excitation wavelength 106 and / or the second excitation wavelength 108 are transmitted by the excitation filter of the excitation spectrum selector 110, such first excitation wavelength 106 and / or second excitation wavelength 108 are directed toward a low-incidence filter 600 positioned to create a small incident angle with respect to the optical path. In certain embodiments, the incident angle of the low-incidence filter 600 can be any incident angle in the range from approximately 10.0° to approximately 30.0°. For example, the incident angle of light with respect to the low-incidence filter 600 can be approximately 10.0°, 11.0°, 12.0°, 13.0°, 14.0°, 15.0°, 16.0°, 17.0°, 18.0°, 19.0°, 20.0°, 21.0°, 22.0°, 23.0°, 24.0°, 25.0°, 26.0°, 27.0°, 28.0°, 29.0°, or 30.0° (but not limited to these).

[0084] At the selected incident angle θ, the low incidence filter 600 is configured to reflect the first excitation wavelength 106 and / or the second excitation wavelength 108 to the objective lens 114 and to permit the transmission of the first emission wavelength signal 116 and / or the second emission wavelength signal 118. The narrow or low incidence filter 600 may be a dichroic, polychroic, short pass, long pass, band pass, band stop, multi-pass, or any desired filter.

[0085] Spectrum edge detection When multiple detection components or phosphors are used in a multi-channel, multi-color fluorescence microscope, spectral overlap or crosstalk can limit the ability to distinguish one detection component signal from another. As disclosed herein, spectral edge detection is a process as a means of distinguishing individual detection components from multiple detection components (e.g., during multiplexing), for example, by orthogonally discriminating the detection components, in that there is no ambiguity as to which detection component is being detected and / or imaged. Improvement in the performance of spectral edge detection can be achieved by minimizing, restricting, and / or reducing to zero optical aberrations in the optical train used in a multi-channel, multi-color fluorescence microscope.

[0086] Regarding some embodiments of the fluorescence microscope described herein, the original image is collected by a light emission detector at a selected wavelength of the emission spectrum. Each original image includes the entire selected emission spectrum signal. Each full signal includes one or more signals from one or more detection components. Each full signal may further include a signal resulting from background or autofluorescence. Regarding spectral edge detection, the characteristics of a signal of interest, e.g., a signal from a detection component of interest, can be identified in the presence of a featureless signal (i.e., a signal having an unknown value and / or structure), e.g., background, autofluorescence, or an unwanted detection component. In other words, spectral edge detection determines the contribution of a detection component of interest in a plurality of signals (or images) composed of contributions from a plurality of detection components having at least partially overlapping spectra by eliminating the contribution from an unwanted detection component in the plurality of signals (or images) when the intensity of the detection component (and thus its respective contribution) is unknown.

[0087] Spectral edge detection should also take into account small-scale variations in the signal, e.g., when detecting a detection component relative to a reference detection component, or when incorporating a detection component that exhibits a luminescence shift based on one or more factors, whether intended (e.g., detecting sample variables including oxygen concentration, metal ion concentration, environmental changes, intracellular uptake (endocytosis), extracellular release (exocytosis), etc.) or unintended (e.g., the detection component exhibits a luminescence shift due to a variable pH of the sample or reagent).

[0088] Spectral edge detection identifies a single detection component within a plurality of spectrally overlapping detection components using the edge (e.g., the rear edge or the front edge) of an emission spectrum curve or an excitation spectrum curve, such as an emission spectrum curve or an excitation spectrum curve for a detection component. For example, two original images can be obtained along the same front or rear spectral edge of a detection component emission spectrum curve, two original images can be obtained along different front and rear spectral edges of a detection component emission spectrum curve, or one original image can be obtained along the front or rear spectral edge of a detection component emission spectrum curve and one original image can be obtained at the peak emission of a detection component emission spectrum curve.

[0089] Spectral edge detection can also utilize combinations of the examples and methods described herein for a single detection component or multiple detection components. For example, when using multiple detection components, the first detection component can be detected by signals from the peak and the front spectral edge, the second detection component can be detected by signals from the front spectral edge, and the third detection component can be detected by signals from the front spectral edge and the rear spectral edge. Further, spectral edge detection can form a curve for the first detection component and a line for the second detection component, such that at least a portion of this line corresponds to a curve that includes data points of each emission spectrum (e.g., signals at a given emission / excitation wavelength).

[0090] FIG. 7A shows the emission spectrum 702 of the first detection component. The emission spectrum 702 includes a front spectral edge (spectral leading edge) 704 and a rear spectral edge (spectral trailing edge) 706. In other words, the front spectral edge 704 is a part of the emission spectrum 702 that is located on the left side of the peak emission 708 or has a wavelength shorter than the peak emission 708, and the rear spectral edge 706 is a part of the emission spectrum 702 that is located on the right side of the peak emission 708 or has a wavelength longer than the peak emission 708. Although the emission spectrum 702 is shown, this spectrum may be an excitation spectrum.

[0091] FIG. 7B shows the emission spectrum 710 of the second detection component. The emission spectrum 710 includes a front spectral edge (spectral leading edge) 712 and a rear spectral edge (spectral trailing edge) 714. In other words, the front spectral edge 712 is a part of the emission spectrum 710 that is located on the left side of the peak emission 716 or has a wavelength shorter than the peak emission 716, and the rear spectral edge 714 is a part of the emission spectrum 710 that is located on the right side of the peak emission 716 or has a wavelength longer than the peak emission 716. Although the emission spectrum 710 is shown, this spectrum may be an excitation spectrum.

[0092] FIG. 7C shows the emission spectrum 720 of the third detection component. The emission spectrum 720 includes a front spectral edge (spectral leading edge) 722 and a rear spectral edge (spectral trailing edge) 724. In other words, the front spectral edge 722 is a part of the emission spectrum 720 that is located on the left side of the peak emission 726 or has a wavelength shorter than the peak emission 726, and the rear spectral edge 724 is a part of the emission spectrum 720 that is located on the right side of the peak emission 726 or has a wavelength longer than the peak emission 726. Although the emission spectrum 720 is shown, this spectrum may be an excitation spectrum.

[0093] In one embodiment, any of a method or system for detecting a dye or detection component while removing background or autofluorescence from an image or signal can be used. For example, two or more original images of a first detection component are provided, such that at least one of these images is located below the lower end of the spectral edge of the first detection component and at least one of these images is located above the upper end of the spectral edge of the first detection component. At least one of the original images includes a signal caused by autofluorescence or background. A first final image of the first detection component is provided, where the first final image is based on the original images from the first detection component and the first final image does not include a signal caused by autofluorescence or background. This can be implemented for any number of detection components to remove background or autofluorescence from any of the images.

[0094] In FIGS. 8A and 8B, a fourth detection component (shown by emission spectrum 804) is used as an example of a method for discriminating individual detection components against background or autofluorescence. However, it should be noted that the methods described herein are not limited to this, and this method can be implemented for the first, second, and / or third detection components (shown by emission spectra 702, 710, 720) or any other suitable detection components.

[0095] FIG. 8A shows an emission spectrum 804 and a background signal 802. The background signal 802 is relatively invariant with respect to the signal of interest and is thus expected to be shown as a constant value (i.e., a straight line) with a known value. In addition, the relative intensities of the emission spectrum 804 and the background signal 802 are unknown.

[0096] For clarity, FIGS. 8B - 9E show images I1 - I 18 obtained from a single wavelength. However, images I1 - I 18It can be obtained over the entire given bandwidth (i.e., 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 75 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm or more), and as a result, the images (shown by the dashed-dotted lines) show the average signal over the entire respective bandwidths. In addition, the original images are obtained, but the signals (e.g., all signals including individual signals) are from the pixels in the original images corresponding to the same locations of the samples between the images. For example, point A is a given location on or within the sample to be imaged. The first pixel representing point A in the first original image includes the first all-signal. The second pixel representing point A in the second original image includes the second all-signal. Using one or more of the methods described herein, the first and second all-signals of the first and second pixels of the first and second original images are evaluated and / or compared to determine the contribution of each detection component.

[0097] FIG. 8B shows original images I1, I2 respectively including first and second signals S1, S2 obtained during imaging. The signals S1, S2 show the total contributions of the fourth detection component and the background 802. The original image I1 includes the first signal S1 on the lower end of the front-side spectral edge, and the original image I2 is taken at the higher end of the front-side spectral edge. To identify the fourth detection component (shown by the emission spectrum 804), the original images I1, I2 are analyzed, and the change in the signals is processed, compared and / or analyzed by any suitable mathematical, computer-calculational, or algebraic process or transformation to determine the relative contribution of the fourth detection component between the first signal S1 and the second signal S2. Such processes or transformations include, but are not limited to, subtraction, differentiation, or combinations thereof. Next, a final image showing the fourth detection component can be provided based on the processing, comparison and / or analysis.

[0098] Spectral edge detection can be performed for each detection component within a plurality of detection components, thereby enabling multiplexing of a sample or a fraction thereof by any desired number of detection components. In one embodiment, at least two detection components can be used for multiplexing. In one embodiment, any suitable number of detection components can be used, the number being 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 30, 32, 40, 50, 60, 70, 80, 90, or 100, but not limited thereto.

[0099] Spectral edge detection can be performed on a detection component having a spectral offset, where the spectral offset is the difference in spectra at comparable spectral edges or at spectral peaks. In one embodiment, this process can be performed on a detection component where the difference in spectral offsets is 50 nm or less. In one embodiment, this process can be performed on a detection component where the difference in spectral offsets is 10 nm or less. In one embodiment, this process can be performed on a detection component where the difference in spectral offsets is 1 to 50 nm or less. In one embodiment, this process can be performed on a detection component where the spectral difference is 10 to 50 nm or less. In one embodiment, this process can be performed on a detection component where the difference in spectral offsets is 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 75 nm, 80 nm, 90 nm, or 100 nm, but is not limited thereto. In one embodiment, the difference between successively positioned spectra (e.g., at the peak) may be the same (e.g., the first detection component and the second detection component are separated by 10 nm, and the second detection component and the third detection component are separated by 10 nm). In one embodiment, the difference between successively positioned spectra (e.g., at the peak) may be different from each other (e.g., the first detection component and the second detection component are separated by 10 nm, and the second detection component and the third detection component are separated by 25 nm).

[0100] In one embodiment, the signal contribution of each detection component (e.g., by contribution or subtraction factor) can be determined by at least two original images, for example, by canceling or zeroing out the signal contribution provided by a detection component of no interest (i.e., when the first detection component is a detection component of no interest and the second detection component is a detection component of interest, and / or also when the first detection component is a detection component of interest and the second detection component is a detection component of no interest) or background / auto-fluorescence. Any number of original images can be obtained for spectral edge detection as long as it is two or more, and such numbers include, but are not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100 or more.

[0101] For clarity with respect to FIGS. 9A - 9E, the emission spectrum 702 of the first detection component is also denoted by "A", the emission spectrum 710 of the second detection component is denoted by "B", and the emission spectrum 720 of the third detection component is denoted by "C". The subscript following A, B, or C in this alias indicates the image in which the contribution intensity of the detection component of interest is. For example, the data point A3 indicates the contribution of A (or the first detection component) in the original image I3 indicated by the data point A3 on the emission spectrum 702. Thus, A3 - A 18 respectively indicate the contribution of A (or the first detection component) in the original images I3 - I 18 , B3 - B 18 respectively indicate the contribution of B (or the second detection component) in the original images I3 - I 18 , and C3 - C 18 respectively indicate the contribution of C (or the third detection component) in the original images I3 - I 18 .

[0102] FIG. 9A shows the emission spectrum 702 of the first detection component and the emission spectrum 704 of the second detection component. The original images I3, I4, I5 are obtained at the designated placement locations on the emission spectrum 702 and the emission spectrum 710. The original image I3 is taken at the lower end of the front spectral edge 704 of the emission spectrum 702, the original image I4 is taken at the higher end of the front spectral edge 712 of the emission spectrum 702, and this higher end also overlaps with the lower end of the front spectral edge 712 of the emission spectrum 710. The original image I5 is taken at the higher end of the front spectral edge 712 of the emission spectrum 710. Although the emission spectra 702, 710 are shown, this spectrum may also be an excitation spectrum.

[0103] In one embodiment, four or more original images can be obtained. In one embodiment, each of the original images is used to analyze only one of the detection components. In one embodiment, at least two (i.e., there is an overlap) of the detection components are analyzed using one or more of the original images. In one embodiment, there are no identical original images among the original images between the first detection component and the second detection component (i.e., all images are separate). In one embodiment, at least one of the original images of the first detection component and at least one of the original images of the second detection component are the same image.

[0104] In one embodiment, the original image taken at the higher end of the rear spectral edge can include the higher end of the front spectral edge, and vice versa (i.e., the original image taken at the higher end of the front spectral edge can include the higher end of the rear spectral edge). In one embodiment, the original image taken at the higher end of a particular spectral edge does not include the higher end of the opposite spectral edge (i.e., the original image at the upper rear spectral edge does not include the upper front spectral edge, and the original image at the upper front spectral edge does not include the upper rear spectral edge).

[0105] To identify the first detection component (shown by the emission spectrum 702) and the second detection component (shown by the emission spectrum 710), the original images I3, I4, I5 are analyzed to determine the relative contributions of the first and second detection components. For example, the relative contributions can be determined by any suitable mathematical, computer computational, or algebraic process or transformation, and such processes or transformations include, but are not limited to, subtraction, differentiation, integration, or combinations thereof. Next, the final image of the first detection component is provided based on the analysis results of the original images I3, I4, for example, the relative contribution of the first detection component over the entire original images I3, I4. Next, the final image of the second detection component is provided based on the analysis results of the original images I4, I5, for example, the relative contribution of the second detection component over the entire original images I4, I5.

[0106] FIG. 9B shows exemplary first and second emission spectra that are substantially the same as the emission spectrum of FIG. 9A, except that the obtained original images I6, I7, I8 are included. The original images I6, I8 are taken at points where the emission intensity of the first detection component has the same or substantially the same value and the emission intensity of the second detection component varies between the images. The original image I7 is taken at a point where at least three data points regarding the second detection component form a line.

[0107] FIG. 9C shows exemplary first and second emission spectra that are substantially the same as the emission spectrum of FIG. 9A, except that the obtained original images I9, I 10 are included. The original image I9 is taken at a point where the higher end of the rear spectral edge of the first detection component overlaps with the lower end of the front spectral edge of the second detection component. The original image I 10is taken at the point where the lower end of the rear spectral edge of the first detection component overlaps with the upper end of the front spectral edge of the second detection component. As shown in FIG. 9C, the rear edge of the first detection component overlaps with the front edge of the second detection component. As a result, the original image I9 includes the upper end of the rear spectral edge of the first detection component and the lower end of the front spectral edge of the second detection component, and the original image I 10 includes the lower end of the rear spectral edge of the first detection component and the upper end of the front spectral edge of the second detection component. In one embodiment, the front edge of the first detection component overlaps with the rear edge of the second detection component.

[0108] FIG. 9D shows exemplary first and second emission spectra that are substantially the same as the emission spectrum of FIG. 9C, except that the obtained original images I 11 ~I 14 are included. The original images I 12 , I 13 are taken at the peaks of the emission spectrum 702 and the emission spectrum 710, respectively. Using the peak emission of the spectra, the relative contributions of the detection components between the original images can be determined, and the other original image is within the spectral edge (front or rear) of the emission spectrum of the detection component from which the peak emission is collected.

[0109] In one embodiment, two or more original images of the first emission spectrum are obtained, where at least one of these images is at the lower end of the spectral edge of the first emission spectrum, and at least one of the images is at the higher end of the same spectral edge of the first emission spectrum. Two or more original images of the second emission spectrum are obtained, where at least one of these images is at the lower end of the spectral edge of the second emission spectrum, and at least one of the images is at the higher end of the same spectral edge of the second emission spectrum. A first final image of the first detection component (indicated by the first emission spectrum) and a second final image of the second detection component (indicated by the second emission spectrum) are provided, where the first and second final images are based on the original images from the first and second detection components. In one embodiment, at least one of the original images of the first and second emission spectra is the same image. For example, the second image of the first emission spectrum (located at the higher end of the spectral edge of the first emission spectrum) is the same image as the first image of the second emission spectrum (located at the lower end of the spectral edge of the second emission spectrum).

[0110] Although two detection components have been described, this process can be used for any number of detection components. In other words, two or more original images of the nth emission / excitation spectrum are obtained, where at least one of these images is at the lower end of the spectral edge of the nth emission / excitation spectrum, and at least one of these images is at the higher end of the spectral edge of the nth emission / excitation spectrum, and n is 1 or greater (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 30, 32, 40, 50, 60, 70, 80, 90, 100 or more). Next, this process is repeated for at least one or more emission / excitation spectra.

[0111] In one embodiment, to determine the signal contribution by individual detection components having overlapping spectra, at least three data points of one detection component should be obtained, as a result of which the three data points form a curve, and at least two data points of another detection component should be obtained, as a result of which the two data points form a line. The determination of which detection component requires data points for forming a curve or data points for forming a line needs to be based on the relative spectral edge. In other words, when using the same spectral edge (i.e., the front side or the rear side) of different emission spectra, an emission spectrum having at least a part of the same spectral edge is classified as an emission spectrum among other emission spectra that require only at least two data points. Using at least two data points (i.e., the data points forming a line), the contribution of the detection component can be determined regardless of the presence or absence of the curve provided by other detection components. Furthermore, using at least three data points (i.e., the data points forming a curve), the contribution of other detection components can be determined regardless of the presence or absence of the line provided by the original detection component.

[0112] In one embodiment, to determine the signal contribution by individual detection components having overlapping spectra, at least three data points of one detection component are collected, as a result of which the three data points form a curve, and at least three data points of another detection component are collected, as a result of which these three data points form a curve or a line. For example, referring back to FIG. 9D and referring to this, the data points of data point B 12 ~B 14 can be substituted into the following equation. TIFF0007712291000001.tif11150In the above equation, C B is the curvature (e.g., the second derivative) of emission B, S B12 is the signal intensity of emission B at the wavelength of image I 12 , S B13 is the signal intensity of emission B at the wavelength of image I 13 , and S B14is the signal intensity of emission B at the wavelength of image I 14 A is the signal intensity of emission B at the wavelength of image I. The data points from A 12 to A 14 can be substituted into the following equation. TIFF0007712291000002.tif11150 In the above equation, C A is the curvature of emission A (e.g., the second derivative), and S A12 is the signal intensity of emission A at the wavelength of image I 12 S is the signal intensity of emission A at the wavelength of image I A13 is the signal intensity of emission A at the wavelength of image I 13 S is the signal intensity of emission A at the wavelength of image I A14 is the signal intensity of emission A at the wavelength of image I 14 The detection components are distinguishable from each other because they have large curvatures (i.e., more positive (e.g., +5 is greater than +2, and as another example, +4 is greater than -1) or more negative (e.g., -6 is greater than -1 in absolute value, and as another example, -5 is greater than +2)) at these emission wavelengths that match the individual detection components. In other words, the detection components have large curvatures across different emission wavelengths. For example, Alexa 647 has a larger curvature than Alexa 594 across the same emission wavelengths from 660 nm, 670 nm, and 680 nm. Alexa 594 has a larger curvature than Alexa 647 across the same emission wavelengths from 609 nm, 619 nm, and 632 nm. Therefore, based on the curvature across 660 nm, 670 nm, and 680 nm, Alexa 674 can be distinguished from Alexa 594, and based on the curvature across 609 nm, 619 nm, and 632 nm, Alexa 594 can be distinguished from Alexa 647.

[0113] Figure 9E shows the three emission spectra 702, 710, 720 (A, B, C) of four images I 15 to I 18 The leading edge of emission B is located below the leading edge of emission A. Therefore, for emission A, at least three data points (e.g., A 15 to A 17Or A 15 , A 16 , A 18 ) is obtained, and for emission B, at least two data points (e.g., B 15 and B 16 , or B 15 and B 17 , or B 16 and B 17 ) are obtained. Additionally, the front edge of emission C is located below the front edge of emission B. Thus, for emission B, at least three data points (e.g., B 15 , B 17 , B 18 or B 16 ~B 18 ) are obtained, and for emission C, at least two data points (e.g., C 16 and C 17 , or C 17 and C 18 , or C 16 and C 18 ) are obtained. Using the respective data points of emissions A~C, the contribution of each detection component can be determined.

[0114] In one embodiment, two or more original images at two different emission wavelengths of a first emission spectrum are obtained, in which case at least one of these images is within the front spectral edge or the rear spectral edge of the first emission spectrum, and at least one of these images is within the rear spectral edge or the front spectral edge of the first emission spectrum. In other words, the two or more original images are within different spectral edges of the same emission spectrum (i.e., at least one original image within the front spectral edge and at least one original image within the rear spectral edge, and the front spectral edge is within the emission spectrum of one detection component).

[0115] As shown in FIG. 9E, the signals of emission spectra A, B are collected via original images on different spectral edges of these emission spectra (for emission spectrum A, A 15 and A 17, for the emission spectrum B, B 15 / B 16 / B 17 and B 18 ). The intensities of the signals on different spectral edges may or may not be equal to each other (for example, the intensity may be large on one spectral edge and small on the other spectral edge).

[0116] In one embodiment, two or more original images of the first emission spectrum are obtained. In this case, at least one of the original images is within the front spectral edge or the rear spectral edge of the first emission spectrum, at least one of these images is within the rear spectral edge or the front spectral edge of the first emission spectrum, and at least one of the original images is in the state of the peak intensity wavelength. In other words, two or more original images are within different spectral edges of the same emission spectrum, and one original image is in the state of the peak intensity wavelength. As shown in FIG. 9E, the emission spectrum A provides signals on different spectral edges of this emission spectrum (for the emission spectrum A, A 15 and A 17 ), and provides a signal in the state of the peak intensity wavelength (for the emission spectrum A, A 16 ).

[0117] FIG. 9E shows the original images collected from the emission spectrum A within the front spectral edge, peak emission, and rear spectral edge, but this is not limited to only one emission spectrum. The original images of the same format can be collected for any desired number of emission spectra.

[0118] In one embodiment, by using the change in signal intensity (for example, pixel level), the detection components can be identified.

[0119] In one embodiment, for example, when a representative point of the emission spectrum is obtained, the rate of change or the change amount of the signal intensity can be determined based on the rear edge of the spectrum. In one embodiment, for example, when a representative point of the emission spectrum is obtained, the rate of change or the change amount of the signal intensity can be determined based on the front edge of the spectrum.

[0120] In one embodiment, for example, when a representative point of the excitation spectrum is obtained, the rate of change or the change amount of the intensity can be determined based on the rear edge of the spectrum. In one embodiment, for example, when a representative point of the excitation spectrum is obtained, the rate of change or the change amount of the intensity can be determined based on the front edge of the spectrum.

[0121] In one embodiment, it is preferable to compare the change amount of the signal intensity with an expected value. For example, the change amount of the intensity may be up to ± (plus or minus) 0.01%, 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the expected value. In one embodiment, it is preferable to compare the change amount of the signal intensity with a threshold value. In one embodiment, the change amount of the signal intensity may be positive or negative, and as a result, the positive or negative change amount identifies the desired detection component.

[0122] In one embodiment, for example, during image processing and analysis, a threshold value can be applied to determine whether the signal is generated by a desired detection component, an undesired detection component, noise, or the background.

[0123] In one embodiment, for example, if the change in signal intensity between a first image and a second image at a desired or predetermined wavelength is greater than or equal to a first threshold, the pixel or signal is "continued" for the resulting analysis image, whereas if the change in signal between the first image and the second image is less than the first threshold, the pixel or signal is "turned off" for the resulting analysis image.

[0124] In one embodiment, a first emission derivative function of the emission spectrum 702 of the first detection component can be obtained, and a second emission derivative function of the emission spectrum 710 of the second detection component can be obtained. Although the first derivative is described, it may be calculated if it is desirable to implement any higher order derivative.

[0125] In one embodiment, for example, if a representative point of the emission spectrum is obtained, the rate of change may be greater than or equal to a threshold. In one embodiment, for example, if a representative point of the emission spectrum is obtained, the change in intensity may be positive, may be positive by at least the amount of the threshold, and / or may be positive by a certain multiple of the first emission. In one embodiment, for example, if a representative point of the excitation spectrum is obtained, the rate of change may be less than or equal to a threshold (i.e., more negative, e.g., -5 is less than -3). In one embodiment, for example, if a representative point of the excitation spectrum is obtained, the change in intensity may be negative, may be negative by at least the amount of the threshold, and / or may be negative by a certain multiple of the first excitation.

[0126] As an example, Δx (change in emission wavelength) is 10 nm, Δy (change in emission intensity) is 50%, and the gradient is 50% / 10 nm or 5% / nm. When comparing the first image and the second image, an increase in intensity of at least 5 times between respective pixels can be attributed to the first detection component, and the pixel is "continued", whereas an increase in intensity of less than 5 times between respective pixels can be attributed to something other than the first detection component (e.g., background), and the pixel is "turned off". This example is not limited to values and / or percentages. The first threshold may include a range based on an expected or anticipated change in emission intensity. For example, the first threshold may be up to a gradient of ± (plus or minus) 0.01%, 0.02%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0127] The gradient satisfies the condition given by the following equation. TIFF0007712291000003.tif12150

[0128] In one embodiment, when the first detection component is excited (thereby obtaining a first image), the wavelength of the first excitation light is preferably selected so as not to excite the second detection component. Next, the wavelength of the second excitation light is also preferably selected so as to excite the first detection component (thereby providing a second image) and not to excite the second detection component. By processing and comparing the first image and the second image, a change in emission intensity (in other words, a gradient, or y / x) can be obtained based on the emission of the first detection component due to the change in the excitation wavelength.

[0129] In one embodiment, at least one of the excitation lights can stimulate one or more detection components. However, the resulting gradient can be used to remove the signals of one or more unwanted detection components, as will be described below.

[0130] In one embodiment, by comparing and processing two or more images resulting from two or more excitation wavelengths with each other, a desired gradient can be calculated. Using the resulting gradient, the signal can be kept on or turned off in the final image. In one embodiment, by comparing and processing two or more signals resulting from two or more excitation wavelengths with each other, a desired gradient can be calculated. The resulting gradient can be used to keep the signal on or turn off the signal in the final image.

[0131] In one embodiment, a first or higher-order derivative can be calculated for each detection component spectral edge. In one embodiment, using the spectral edges of the respective detection components, the emissions of different detection components can be distinguished from each other.

[0132] In one embodiment, the minimum number of original images is n, where n is the number of detected components. For example, the first original image can be obtained above the upper end of the rear edge of the first emission spectrum and below the lower end of the front edge of the second emission spectrum. The second original image can be obtained below the lower end of the rear edge of the first emission spectrum and above the upper end of the front edge of the second emission spectrum. Processing and / or analyzing the first and second original images can provide a first final image of the first detected component (indicated by the first emission spectrum) and a second final image of the second detected component (indicated by the second emission spectrum). Although emission spectra are described, this embodiment can be implemented for excitation spectra.

[0133] In one embodiment, the minimum number of original images is n + 1, where n is the number of detected components.

[0134] In one embodiment, all of the original and final images of the first and second detected components are displayed to a end user or operator, for example, on a screen (e.g., at least one screen of a phone, tablet, computer, television, PDA, handheld device, etc.). In one embodiment, at least one of the original images of the first and / or second detected components is displayed. In one embodiment, at least one of the final images of the first and / or second detected components is displayed. In one embodiment, none of the original images are displayed, but at least one of the final images is displayed. In one embodiment, none of the original images are displayed, but all of the final images are displayed.

[0135] Examples of collecting signals (e.g., the same spectral edge, different spectral edges from each other, a peak and one spectral edge, a peak and two spectral edges, etc.) are not limited to the emission spectra specifically described for the exemplary collection. Rather, signal collection is applicable to one or more emission spectra, where all emission spectra have the same collection (e.g., the same spectral edge, different spectral edges from each other, a peak and one spectral edge, a peak and two spectral edges, etc.), at least two emission spectra have the same collection, or there is no emission spectrum having the same collection.

[0136] To obtain the original image, imaging is preferably performed using a flow cytometer or a microscope, such as a fluorescence microscope, a scanner, etc. Imaging can be performed using conventional epi-fluorescence, light sheet microscope, super-resolution microscope, and confocal microscope.

[0137] Regardless of whether it is an image or a file, whether it is an original image or a processed one, it can be stored in any appropriate storage medium at any time when implementing any embodiment of the present invention. Examples of the storage medium include, but are not limited to, hard disks, random access memory (RAM), read-only memory (ROM), storage devices of distributed computing systems, optical disks (e.g., compact disks, digital versatile disks, or Blu-ray (registered trademark) disks), flash memory devices, memory cards, etc.

[0138] Embodiments of the present invention include non-transitory computer-readable media that can store instructions for implementing the above-described methods and any of their steps (including any combination of the methods and steps). For example, the non-transitory computer-readable media can store instructions executable by one or more processors or similar devices.

[0139] Embodiments of the present invention include two or more non-transitory computer-readable media capable of storing instructions for performing the above-described methods and any steps of these methods (which include any combination of methods and steps). For example, the instructions for execution can be divided among two or more processors or similar devices.

[0140] Another embodiment of the present invention further includes a computer or device (e.g., a telephone, a tablet, a PDA, etc.) that reads and executes computer-executable instructions, for example, a non-transitory computer-readable medium recorded or stored on a storage medium (which may be the same as or different from the storage medium for storing images or files as described above) for performing the functions of any embodiment. The computer can include one or more of a central processing unit (CPU), a microprocessor unit (MPU), or other circuit configurations, and can also include a network of separate computers or separate computer processors. The computer-executable instructions can be provided to the computer, for example, from a network or a storage medium.

[0141] The computer or device may also be configured to display any of the images or files, whether the original image or the processed image, for example, on a monitor or a screen.

[0142] If a feature or element is described herein as being located "above" another feature or element, this may mean that it is located directly above the other feature or element, or that intervening features or elements may also be present. In contrast, if a feature or element is described as being located "directly above" another feature or element, there are no intervening features or elements. Also, if a feature or element is described as being "connected", "attached", or "coupled" to another feature or element, this may mean that it is directly connected, attached, or coupled to the other feature or element, or that intervening features or elements may be present. In contrast, if a feature or element is described as being "directly connected", "directly attached", or "directly coupled" to another feature or element, there are no intervening features or elements. Although an embodiment has been described or illustrated, the features or elements described or illustrated apply to other embodiments. Also, as would be understood by one of ordinary skill in the art, reference to a structure or feature provided "adjacent" to another feature may have portions that overlap or are located beneath the adjacent feature.

[0143] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, the singular forms "a", "an", and "the" used in the original specification include the plural forms as well unless the context clearly dictates otherwise. Further understood, the terms "comprises" and / or "comprising", as used herein, specify the presence of the stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. The expression "and / or" as used herein includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / " in some cases.

[0144] Spatially, graphically, or numerically related terms, such as "under", "below", "beneath", "above", "over", "higher than", etc., may be used in this specification for convenience of explanation to describe the relationship of one element or feature to another element or feature as shown in the drawings. As understood, spatially related terms are intended to include different orientations in use or operation in addition to the orientation shown in the drawings. For example, if the illustrated device, system, or direction is reversed, an element described as "under" or "beneath" another element or feature will face "above" the other element or feature. Thus, the exemplary term "under" may include both upward and downward orientations. Or, the device may be oriented in another direction (rotated 90° or other orientations), and the spatially related descriptions used in this specification will be interpreted accordingly. Similarly, "above", "below", "perpendicular to", "horizontal to", etc. are used in this specification for purposes of explanation unless otherwise specified. Further, "under", "higher than", etc. are used to indicate elements, features, information, etc., which are located further down or further up in a table, graph, or plot, or are smaller or larger in value or intensity.

[0145] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless otherwise specified. These terms may be used to distinguish one feature / element from another. Thus, the first feature / element described below may be referred to as the second feature / element, and similarly, the second feature / element described below may be referred to as the first feature / element, without departing from the teachings of the present invention. Additionally, although the terms "first" and "second" are used, they are not intended to limit the various features / elements to only one or two. Rather, they may be included or used when three (i.e., a third), four (i.e., a fourth), or more are applicable or desirable.

[0146] Throughout the specification and the following claims, unless otherwise required, the word "comprise" and its variations, such as "comprises" and "comprising", are meant to imply that various components can be used jointly in a method and an article (e.g., a composition and an apparatus including a device and a method). For example, the term "comprising" is understood to suggest including any recited element or step, but not to mean excluding any other element or step.

[0147] In this specification and the claims, unless otherwise expressly specified, all numbers used in the examples should be read as if the word "about" or "substantially" were positioned before the word, even if this term does not explicitly appear. The phrases "about" or "substantially" may be used when describing magnitudes and / or positions to indicate that the recited values and / or positions are within a reasonable expected range of values and / or positions. For example, a numerical value may have a value of ±0.1% of the recited value (or range of values), ±1% of the recited value (or range of values), ±2% of the recited value (or range of values), ±5% of the recited value (or range of values), ±10% of the recited value (or range of values), etc. Any numerical value given in this specification should also be understood to include about or substantially that value unless otherwise specified. For example, if the value "10" is shown, "about 10" is also shown. Any numerical range recited in this specification is intended to include all sub-ranges subsumed therein. Also, as will be appropriately understood by those skilled in the art, when a value is shown, it goes without saying that "less than the value", "greater than or equal to the value", and the conceivable ranges between these values are also shown. For example, if the value "X" is shown, "less than X", as well as "greater than or equal to X" (for example, X is a numerical value) are also shown. Also, throughout this application, data is provided in many different formats, and this data represents ranges by any combination of endpoints and starting points and data points. For example, if a particular data point "10" and a particular data point "15" are shown, it goes without saying that greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, equal to 10, as well as greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, equal to 15 are disclosed and considered to be 10 - 15. Also, it goes without saying that each unit between two particular units is also disclosed. For example, if 10 and 15 are shown, 11, 12, 13, 14 are also shown.

[0148] Although various exemplary embodiments have been described above, any of a number of modifications can be made to the various embodiments without departing from the scope of the invention as recited in the claims. For example, the order in which the various described method steps are performed is often changed in alternative embodiments, and in other alternative embodiments, one or more method steps may be omitted entirely. There are embodiments that include features as options of the various apparatus and system embodiments, and there are also embodiments that do not include them. Accordingly, the above description is primarily for illustrative purposes and should not be construed as limiting the scope of the invention as if the scope of the invention were recited in the claims.

[0149] The examples and instances included in this specification are for illustrative purposes only and not for the purpose of limitation, and show specific embodiments for carrying out the subject matter of the invention. As described above, other embodiments can be utilized and derived from specific embodiments, and as a result, structural and logical substitutions and changes can be made without departing from the scope of the invention. It is merely for convenience and is not intended to spontaneously limit the scope of the invention to any single invention or the technical idea of the invention. In fact, when two or more are disclosed, such embodiments of the subject matter of the invention may be referred to herein individually or collectively by the expression "the invention". Thus, although specific embodiments have been illustrated and described in this specification, configurations devised to achieve the same purpose can be used in place of the specific embodiments illustrated. The present disclosure includes any and all modifications or variations of the various embodiments. The combinations of the above-described embodiments and other embodiments specifically described in this specification will be apparent to those skilled in the art upon consideration of the above description.

[0150] The above description has used a specific terminology system for illustrative purposes to provide a thorough understanding of the present invention. However, as will be apparent to those skilled in the art, specific details are not necessarily required to implement the systems and methods described herein. The above description of specific embodiments has been provided illustratively for purposes of illustration and description. The above description is not intended to be exhaustive or to limit the disclosure to the forms described. Many modifications and variations are possible in light of the above teachings. Embodiments are illustrated and described in order to best explain the principles and practical applications of the disclosure, thereby enabling those skilled in the art to make various embodiments with various modifications suitable for the particular uses contemplated by the disclosure and to make optimal use of them. The scope of the present invention is defined by the claims below and their equivalents.

Claims

**Claim 1** An imaging system, including at least one tiltable filter assembly including an optical filter configured to be positioned in the optical path of the imaging system, a telecentric tube lens configured to be positioned in the optical path of the imaging system at a position where the telecentric tube lens is telecentric in both the image and object spaces, a dynamic correction optical system configured to be positioned in the optical path of the imaging system, and a fixed correction optical system configured to be positioned in the optical path of the imaging system, wherein the fixed correction optical system is configured to reduce the aberration generated by the combination of the optical filter and the dynamic correction optical system. An imaging system. **Claim 2** The imaging device according to claim 1, wherein even if residual aberration is generated by the combination of the fixed correction optical system, the dynamic correction optical system, and the optical filter, the residual aberration is zero (0). **Claim 3** Regarding a selected first incident angle of the optical filter, the dynamic correction optical system is configured to be tilted up to a second incident angle, and the incident angle of the fixed correction optical system is the optical filter at the selected first incident angle. The imaging system according to claim 1, wherein the imaging system is configured to generate an aberration for substantially reducing the aberration generated by the combination of the dynamic correction optical system at the second incident angle. **Claim 4** The imaging system according to claim 1, wherein the dynamic correction optical system is configured to substantially stabilize the lateral image shift generated by the tilt of the optical filter. **Claim 5** The imaging system according to claim 4, wherein the sum of the lateral image shift generated by the dynamic correction optical system and the lateral image shift generated by the tilt of the optical filter is substantially constant. **Claim 6** Regarding a selected first incident angle of the optical filter, the dynamic correction optical system is configured to be tilted up to a second incident angle, and the sum of the lateral image shift generated by the dynamic correction optical system and the lateral image shift generated by the optical filter is substantially constant. The imaging system according to claim 4. **Claim 7** The optical filter held by the at least one tiltable filter assembly and the dynamic correction optical system are configured to tilt on a parallel X axis, and the fixed correction optical system is tilted on a Y axis perpendicular to the X axis. The imaging system according to claim 1.

8. The imaging system according to claim 1, further comprising a filter changer configured to hold the at least one tiltable filter assembly.

9. The filter changer is a filter wheel, and the at least one tiltable filter assembly is configured to tilt the optical filter to an incident angle selected from the range of 0° to 89.9°. The imaging system according to claim 8.

10. The filter changer includes at least two tiltable filter assemblies. The imaging system according to claim 8.

11. The imaging system according to claim 1, further comprising a low-incidence filter selected from at least one of the group consisting of a dichroic filter, a polychroic filter, a short-pass filter, a long-pass filter, a band-pass filter, a band-stop filter, and a multi-pass filter, and the low-incidence filter is configured to have an incident angle of excitation light selected from an angle in the range of 10.0° to 30.0°.

12. The low-incidence filter is a polychroic filter. The imaging system according to claim 11.

13. The imaging system is a fluorescence microscope type imaging system. The imaging system according to claim 1.

14. The optical filter is an interference filter. The imaging system according to claim 1.

15. The at least one tiltable filter assembly is configured to tilt the optical filter to an incident angle selected from the range of 0° to 89.9°. The imaging system according to claim 1.

16. A method for reducing the aberration of an imaging system, the method comprising: Positioning an optical filter in the optical path of the imaging system at a first incident angle; Positioning a dynamic correction optical system in the optical path of the imaging system at a second incident angle; And positioning a fixed correction optical system in the optical path of the imaging system at a third incident angle. The incident angle of the fixed correction optical system is configured to generate an aberration for substantially reducing the astigmatism of the optical filter at the first incident angle and the overall astigmatism of the dynamic correction optical system at the second incident angle. Method. **Claim 17** A method for stabilizing a lateral image shift of an imaging system, the method comprising: positioning an optical filter in the optical path of the imaging system at a first incident angle; positioning a telecentric tube lens in the optical path of the imaging system at a position where the telecentric tube lens is telecentric in both the image and object spaces; positioning a dynamic correction optical system in the optical path of the imaging system at a second incident angle, the dynamic correction optical system being configured to substantially stabilize a lateral image shift caused by tilting of the optical filter. Method.

Images