Imaging chain and spectral edge detection for an imaging system

Abstract

The present disclosure relates generally to an imaging train for an imaging system. More specifically, the present disclosure relates to an imaging system configured to limit optical aberrations. Furthermore, the present disclosure relates to a method of limiting optical aberrations in an imaging system configured for imaging a sample, but more specifically for detecting individual detection moieties within a plurality of detection moieties.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 005,932, filed April 6, 2020, the entire contents of which are incorporated herein by reference.

[0002] Reference Citation

[0003] The entire contents of all publications and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. TECHNICAL FIELD

[0004] The present disclosure relates generally to microscopy imaging systems for analyzing samples. More specifically, the present disclosure relates to mitigating optical aberrations of an imaging system for a fluorescence microscope. Further, the present disclosure relates to imaging trains for automating high-throughput imaging systems. Additionally, the present disclosure relates to methods and imaging systems for sequentially detecting multiple detection moieties. BACKGROUND

[0005] Microscopes are a powerful technology for inspecting and imaging inorganic and organic samples. Selected samples can include biological samples having one or more biomarkers or components that can be targeted for detection or imaging. Current imaging train filters and imaging systems for microscopes can only permit a limited number of labels to be used at any given time. Further, imaging systems can encounter image quality issues such as aberrations, astigmatism, focus offset, and image offset. Accordingly, practitioners, researchers, and microscopy technicians have continually sought systems and methods for more efficiently and accurately imaging samples. SUMMARY

[0006] In a first aspect, an embodiment of an imaging system is provided. The imaging system includes at least one tiltable filter assembly including an optical filter configured to be disposed in an optical path of the imaging system, a dynamic correction optic configured to be disposed in the optical path of the imaging system, and a fixed correction optic configured to be disposed in the optical path of the imaging system.

[0007] The imaging system can include a telecentric tube lens configured to be disposed in the optical path of the imaging system.

[0008] In some embodiments, the fixed correction optic is configured to substantially mitigate astigmatism caused by a combination of the optical filter and the dynamic correction optic.

[0009] Any residual astigmatism caused by the combination of the fixed correction optics, the dynamic correction optics, and the optical filter can be approximated to zero (0).

[0010] In some embodiments, for a selected first angle of incidence of the optical filter, the dynamic correction optics is configured to tilt to a second angle of incidence, and wherein the angle of incidence of the fixed correction optics is configured to produce astigmatism to substantially mitigate astigmatism caused by the combination of the optical filter at the selected first angle of incidence and the dynamic correction optics at the second angle of incidence. The dynamic correction optics can be configured to substantially stabilize lateral image shift caused by tilting of the optical filter. In some embodiments, the sum of lateral image shift caused by the dynamic correction optics and lateral image shift caused by tilting of the optical filter is approximately constant. For a selected first angle of incidence of the optical filter, the dynamic correction optics can be configured to tilt to a second angle of incidence, and wherein the sum of lateral image shift caused by the dynamic correction optics and lateral image shift caused by the optical filter is approximately constant.

[0011] In some embodiments, the optical filter and the dynamic correction optics clamped by the at least one tiltable filter assembly are configured to tilt in a substantially parallel X-axis, and wherein the fixed correction optics tilts in a substantially perpendicular Y-axis. The telecentric tube lens can be located at a position in the optical path of the imaging system where the telecentric tube lens is telecentric in both image space and object space.

[0012] In some embodiments, the system includes a filter translator configured to clamp the at least one tiltable filter assembly. The filter translator can be a filter wheel, and the at least one tiltable filter assembly can be configured to tilt the optical filter to an angle of incidence selected from a range of substantially 0° to 89.9°. In some embodiments, the filter translator includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tiltable filter assemblies.

[0013] In some embodiments, the system includes a low incidence filter selected from at least one of the group consisting of a dichroic filter, a multichroic filter, a short pass filter, a long pass filter, a band pass filter, a band reject filter, and a multi-pass filter, wherein the low incidence filter is configured to have an angle of incidence of excitation light selected from a range of substantially 10.0° to 30.0°. The low incidence filter can be a multichroic filter.

[0014] In some embodiments, the imaging system is a fluorescence microscope imaging system.

[0015] The optical filter can be an interference filter.

[0016] In some embodiments, the at least one tiltable filter assembly is configured to tilt the optical filter to an angle of incidence selected from the range of substantially 0° to 89.9°.

[0017] In another aspect, a method of mitigating astigmatism in an imaging system is provided. The method includes the steps of: disposing an optical filter in an optical path of the imaging system at a first angle of incidence; disposing a dynamic correction optic in the optical path of the imaging system at a second angle of incidence; disposing a fixed correction optic in the optical path of the imaging system at a third angle of incidence; wherein the angle of incidence of the fixed correction optic is configured to produce astigmatism to substantially mitigate a combined astigmatism of the optical filter at the first angle of incidence and an astigmatism of the dynamic correction optic at the second angle of incidence.

[0018] In yet another aspect, a method of stabilizing lateral image shift in an imaging system is provided. The method includes the steps of: disposing an optical filter in an optical path of the imaging system at a first angle of incidence; disposing a dynamic correction optic in the optical path of the imaging system at a second angle of incidence; wherein the dynamic correction optic is configured to substantially stabilize a lateral image shift caused by a tilt of the optical filter. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 FIG. 1 is a schematic diagram illustrating an embodiment of an imaging chain for a fluorescence microscope imaging system.

[0020] Figure 2A FIG. 2 is a perspective view of one embodiment of a filter wheel.

[0021] Figure 2B FIG. 3 is a perspective view of one embodiment of a filter assembly.

[0022] Figure 3 FIG. 4 is a perspective view of one embodiment of a filter assembly, cam, and motor.

[0023] Figure 4A FIG. 5 is a schematic diagram illustrating one embodiment of an imaging chain for a fluorescence microscope imaging system.

[0024] Figure 4B FIG. 6 is a perspective view of selected elements of an imaging chain for a fluorescence microscope imaging system.

[0025] Figure 5 FIG. 7 is a schematic diagram illustrating another embodiment of an imaging chain for a fluorescence microscope imaging system.

[0026] Figure 6 FIG. 17 is a graph showing emission spectra of a first detection moiety.

[0027] Figure 7A FIG. 18 is a graph showing emission spectra of a second detection moiety.

[0028] Figure 7B FIG. 19 is a graph showing emission spectra of a third detection moiety.

[0029] Figure 7C FIG. 20 is a graph showing emission spectra and background signal.

[0030] Figure 8A FIG. 21 is a graph showing emission spectra and imaging.

[0031] Figure 8B Signal obtained during imaging.

[0032] Figures 9A to 9D FIG. 23 is a graph showing emission spectra and imaging of a first detection moiety and a second detection moiety.

[0033] Figure 9E FIG. 24 is a graph showing emission spectra and imaging of a first detection moiety, a second detection moiety, and a third detection moiety. DETAILED DESCRIPTION

[0034] In the following description, the term“raw image” is used to describe an image that includes data or at least one signal that has been captured by a sensor or detector, that has not been processed, whether or not visually displayed to an operator or end user.

[0035] In the following description, the term“final image” is used to describe an image that includes data or at least one signal that has been processed, whether or not visually displayed to an operator or end user. “Final image” can also be used to describe an image that is an output image resulting from a comparison and / or analysis of two or more raw or other final images, whether or not visually displayed to an operator or end user.

[0036] In the following description, the term“light” is not intended to be limited to describing electromagnetic radiation in the visible portion of the electromagnetic spectrum, but is also intended to describe radiation in the ultraviolet portion and the infrared portion of the electromagnetic spectrum.

[0037] In the following description, the term“sample” is used to describe an organic solid, an organic fluid, an inorganic solid, an inorganic fluid, a biological fluid, a biological semi-solid, a biological solid (which can hold a solid such as a tissue, or which can be liquefied in any appropriate manner), a suspension, a portion of a suspension, a component of a suspension, and the like.

[0038] In the following description, the term "target analyte" or "target material" is used to describe the biological material of interest.

[0039] In the following description, the term "non-target analyte" is used to describe biological material that is not the target analyte.

[0040] In the following description, the term "biomarker" is used to describe a substance present on or within the target analyte or target material (i.e., intracellular or extracellular to the target analyte; such as internalized within the target analyte by phagocytosis; or the like). Biomarkers include, but are not limited to, peptides, proteins, subunits, domains, motifs, epitopes, isoforms, DNA, RNA, and the like. Biomarkers can be target molecules for drug delivery.

[0041] In the following description, the term "affinity molecule" is used to describe any molecule capable of binding or interacting with another molecule. The interaction or binding can be covalent or non-covalent. Affinity molecules include, but are not limited to, antibodies, haptens, proteins, aptamers, oligonucleotides, polynucleotides, or any suitable molecule for interacting or binding with another molecule (e.g., a biomarker; a binding pair molecule or a complementary molecule, including but not limited to biotin or avidin; or the like).

[0042] In the following description, the term "channel" is used to describe a color or range of colors based on the color of the signal provided by one or more detection groups of a fluorophore. The color or range of colors is obtained based on the selected interference filter (i.e., excitation filter, emission filter, polychromatic filter, dichromatic filter) and / or the wavelength of the signal. For example, a channel can be purple, blue, green, yellow, orange, red, deep red, etc. Further, when multiple channels are used, each channel has a specific color or range of colors. For example, a first channel can be green and a second channel can be orange. It should be noted that although two or more detection groups can provide signals having different wavelengths, the signals can be in the same channel based on the filter set used. For example, a first detection group provides a signal having a wavelength of 488 nm and a second detection group provides a signal having a wavelength of 500 nm. Even though the wavelengths are not the same, the filter set in one channel passes both wavelengths of 488 nm and 500 nm to allow both to be imaged simultaneously, thereby producing a single image that includes both 488 nm and 500 nm emissions. A channel can also describe an excitation and emission bandpass combination.

[0043] In the following description, the term "detection group" is used to describe a compound or substance that provides a signal for detection, thereby indicating the presence of another compound or substance, analyte, etc. within a sample or specimen. The detection group can be fluorescent (such as a fluorescent probe) or chromogenic (such as a chromogenic dye). The fluorescent probe can be a reactive dye, an organic dye, a fluorescent protein, a quantum dot, a non-protein organic molecule, a nanoparticle (e.g., nanodiamond), a phosphor integrated dot (PID), etc.

[0044] A detection group is a compound or substance that provides a signal for detection, thereby indicating the presence of another compound or substance, analyte, etc. within a sample or specimen. The detection group can be used as a tracer, a label for certain structures, a label for a biomarker, etc. The detection group can be distributed or can label an appropriate structure or biomarker in a manner including, but not limited to, uptake, selective uptake, diffusion, and attachment to a linking molecule. The detection group can be bound to a biomarker by direct labeling or indirect labeling.

[0045] 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-benzidine dihydrochloride / ortho-phenylenediamine (Hanker-Yates reagent), Fast Red TR, New Fuchsin, Fast Blue BB, etc. Fluorescent probes include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthylfluorescein; 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 Fuchsin; ACMA (9-amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavine; Acriflavin Feulgen SITSA; Aequorin (Luminescent Protein); Autofluorescent Protein; Alexa Fluor 350 TM ; Alexa Fluor 430 TM ; Alexa Fluor 488 TM ; Alexa Fluor 532 TM ; Alexa Fluor 546 TM ; Alexa Fluor 568 TM; Alexa Fluor 594 TM ; Alexa Fluor 633 TM ; Alexa Fluor 647 TM ; Alexa Fluor 660 TM ; Alexa Fluor 680 TM ; Alizarin Complexone; Alizarin Red; Allophycocyanin (APC); AMC; AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminoglycoside; Aminoglycoside D; Aminocoumarin; Aminomethylcoumarin (AMCA); Aniline Blue; Anthracene Stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG TM CBQCA; ATTO-TAG TM FQ; Auramine; Aurone G; Aurone; BAO9 (Bisaminophenyl Oxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulfate; Beta Lactamase; BFP Blue Shift GFP (Y66H; Blue Fluorescent Protein); BFP / GFP FRET; Bimane; Bisbenzamide; Bisbenzimidazole (Hoechst); BisBTC; Blankophor FFG; Blankophor SV; BOBO TM -1; BOBO TM -3; Borofluorodipyrromethene 492 / 515; Borofluorodipyrromethene 493 / 503; Borofluorodipyrromethene 500 / 510; Borofluorodipyrromethene 505 / 515; Borofluorodipyrromethene 530 / 550; Borofluorodipyrromethene 542 / 563; Borofluorodipyrromethene 558 / 568; Borofluorodipyrromethene 564 / 570; Borofluorodipyrromethene 576 / 589; Borofluorodipyrromethene 581 / 591; Borofluorodipyrromethene 630 / 650-X; Borofluorodipyrromethene 650 / 665-X; Borofluorodipyrromethene 665 / 676; Borofluorodipyrromethene Fl; Borofluorodipyrromethene FL ATP; Borofluorodipyrromethene Fl-Ceramide; Borofluorodipyrromethene R6GSE; Borofluorodipyrromethene TMR; Borofluorodipyrromethene TMR-X conjugate; Borofluorodipyrromethene TMR-X, SE; Borofluorodipyrromethene TR; Borofluorodipyrromethene TR ATP; Borofluorodipyrromethene TR-X SE; BO-PRO TM -1; BO-PRO TM -3; Brilliant Sulphanthine 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; Calcofluor White TM; 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 TM ; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (cyan fluorescent protein); CF405S; CF488A; CF 488; CF543; CF 647; CF 750; CF 760; CF 780; FP / YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin- Lucifer Yellow; C-Phycocyanin; CPM Methyl Coumarin; CTC; CTC Methyl Coumarin; Cy2 TM ; Cy3.1 8; Cy3.5 TM ; Cy3 TM ; Cy5.1.8; Cy5.5 TM ; Cy5 TM ; Cy7 TM ; Cyan GFP; Cyclic AMP Fluorescent Sensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Dansyl; Dansylamine; Dansyl Chloride; Dansyl DHPE; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (dichlorodihydrofluorescein diacetate); DDAO; DHR (dihydro rhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS; DiA (4-Di-16-ASP); Dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (DiIC18(5)); DIDS; Dihydro rhodamine 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); ELF 97; Eosin; ER-Tracker TM Green; ER-Tracker TM Red; ER-Tracker TMBlue-white DPX; Erythrosine; Erythrosine ITC; Ethidium bromide; Ethylphenidium bromide dimer-1 (EthD-1); Euflavin; EukoLight; Europium(III) chloride; EYFP (Enhanced Yellow Fluorescent Protein); Solid Blue; FDA; FIF (Formaldehyde-Induced Fluorescence); FITC; FITC antibody; Frazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluoroemerald; Fluorogold (Hydroxystilbene); Fluororuby; Fluorine X; FM1-43 TM FM 4-46; Pyrimidine Red TM (High pH); Pyrimidine Red TM / Pyrimidine-3; Pyrimidine-2, high calcium; Pyrimidine-2, low calcium; Pyrimidine-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Powder 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red-shifted (rsGFP); GFP wild-type, non-UV excitation (wtGFP); GFP wild-type, UV excitation (wtGFP); GFPuv; Hydroxamic 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); Intrawhite Cf JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucoflavous Brightener PAF; Leucoflavous Brightener SF; Leucoflavous Brightener WS; Lissamine rhodamine; Lissamine rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer yellow; LysoTracker Blue; Lyso Tracker Blue / White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Magdala red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium green; Magnesium orange; Malachite green; Marina blue; Maxilon Brilliant Flavine 10GFF; Maxilon Brilliant Flavine 8GFF; Merocyanin; Methoxycoumarin; Mitotracker Green; Mitotracker Orange; Mitotracker Red; Monacolin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (methyl green pyronine styryl); mStrawberry; NBD; NBD amine; Nile red; Nitrobenzoxadiazone; Noradrenaline; Nuclear fast red; Nuclear yellow; Nylosan Brilliant Lavin E8G; Oregon green TM 488; Oregon green TM 500; Oregon green TM 500; Oregon green TM514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B; Phycoerythrin R; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Proxyl Yellow; Propidium Iodide (PI); Pyrene; Pyronine; Pyronine 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; QSY 7; Quinacrine Mustard; Red 613 (P E Texas Red); Resorcinol; RFP; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-Phyco cyanin; 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 TM(Superluminescent GFP; SITS (Primuline); SITS (Stilbene isothiosulfonate); SNAFL Calcein; SNAFL-1; SNAFL-2; SNARF Calcein; SNARF1; Sodium Green; Chromatographic Light Green; Chromatographic Green; Chromatographic Orange; Chromatographic Red; SPQ (6-Methoxy-N-(3-Sulfopropyl)quinoline); Stilbene; Sulfarodamine B can C; Sulfarodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; SYTOX Red; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red TM Texas Red-X TM Couplings; Thiocarbonylcyanine (DiSC3); Thiazide Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Sulfur Electrolyte; 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; True Blue; TruRed; Tubulin Tracker TMGreen; Ultralite; Uranine B; Uvitex SFC; wt GFP (wild-type GFP); WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP (Yellow Shift); 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-675, DY-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. In one embodiment, a detection moiety such as an organic fluorophore can have a molecular weight of substantially 100 daltons or greater, including but not limited to at least 1 kilodalton, at least 10 kilodaltons, at least 25 kilodaltons, at least 50 kilodaltons, at least 75 kilodaltons, at least 100 kilodaltons, at least 150 kilodaltons, at least 200 kilodaltons, at least 250 kilodaltons, at least 300 kilodaltons, at least 340 kilodaltons, at least 350 kilodaltons, at least 500 kilodaltons, and at least 750 kilodaltons.

[0046] In the following description, the terms "stain" or "label" are used interchangeably to describe an affinity molecule that binds or interacts with a detection group. The binding or interaction can be direct or indirect. Direct binding or interaction includes covalent or non-covalent interaction between a biomarker and a detection group. Indirect binding or interaction includes the use of at least a first complementary molecule and a second complementary molecule that form a binding pair. The first and second complementary molecules combine to form a binding pair that can bind or interact in at least one of the following ways: hydrophobic interaction, ionic interaction, hydrogen bonding interaction, non-covalent interaction, covalent interaction, affinity interaction, and the like. Binding pairs include, but are not limited to: immuno-type binding pairs such as antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or first antibody-second antibody; non-immuno-type binding pairs such as biotin-avidin, biotin-streptavidin, folate-folate binding protein, hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme-substrate analog, enzyme-pseudosubstrate (substrate analog that cannot be catalyzed by enzyme activity), enzyme-cofactor, enzyme-regulator, enzyme-inhibitor, or vitamin B12-intrinsic factor. 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; polymeric linker (e.g., polyethylene glycol); or polynucleotide-polynucleotide binding protein. Binding pairs can be included within or used as amplification techniques. Amplification techniques are also implemented to increase the number of detection groups that bind or interact with the biomarker to increase the signal. In one embodiment, when a binding pair is used, the stain can be pre-bound such that during the labeling, staining, or adding step, the affinity molecule is already bound or interacted with the detection group when added to the sample. In one embodiment, when a binding pair is used, the stain can be conjugated in the sample such that the labeling, staining, or adding step includes introducing (in any desired or appropriate order) an affinity molecule-first binding molecule conjugate and a second binding pair molecule-detection group conjugate, where the first and second binding pair molecules are complementary and bind or interact with each other.

[0047] Further, "a plurality of stains" can be used to describe two or more stains where the affinity molecules and / or detection groups are different. 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.

[0048] In the following description, the term "conjugate" is used to describe a first chemical, molecule, moiety, etc. that is bound to or interacts with a second chemical, molecule, moiety, etc. The binding or interaction is direct or indirect. Direct binding or interaction includes covalent or non-covalent interaction between a biomarker and a detection moiety. Indirect binding or interaction includes the use of at least a first complementary molecule and a second complementary molecule that form a binding pair. The first and second complementary molecules combine to form 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. Binding pairs include, but are not limited to: immunological binding pairs such as antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or first antibody-second antibody; non-immunological binding pairs such as biotin-avidin, biotin-streptavidin, folate-folate binding protein, hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme-substrate analog, enzyme-pseudosubstrate (substrate analog that cannot be catalyzed by enzyme activity), enzyme-cofactor, enzyme-regulator, enzyme-inhibitor, or vitamin B12-intrinsic factor. 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; polymeric linker (e.g., polyethylene glycol); or polynucleotide-polynucleotide binding protein.

[0049] In the following description, the term "signal" is used to describe an electrical current or electromagnetic field that carries data from one place or source to another place or detector. For example, a signal can be light emitted by a detection moiety to convey the presence of a detection moiety on or within a sample, such as a cell, or a target analyte.

[0050] In the following description, the term "multiplexing" is used to describe a process or kit that labels a sample with multiple stains. Each of the detection moieties emits a different wavelength. For example, at least two stains can be used to label a sample. Multiplexing can include up to 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100, or more stains.

[0051] Example methods for labeling biomarkers on target analytes are discussed. In one embodiment, a sample suspected of including at least one target analyte is obtained. Suitable apparatus, systems, and / or methods for sample collection and / or processing can include those described in one or more of the following U.S. patents and published applications, each of which is hereby incorporated by reference in its entirety: 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; 2014 / 0161688; 2017 / 0014819; 2017 / 0059552; 2017 / 0074759; 62 / 873,390. Suitable apparatus, systems, and / or methods for target analyte recovery, isolation, or selection can include those described in one or more of the following U.S. patents and published applications, each of which is hereby incorporated by reference in its entirety: 9,222,953; 9,440,234; 9,519,002; 9,810,605; 2017 / 0219463; 2017 / 0276575.

[0052] In one embodiment, a sample can be subjected to staining after collection and or processing. In one embodiment, a sample can be multiplexed. At least one stain is added to the sample to be labeled, such as by an automated stainer 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 material is stained. In one embodiment, at least one target analyte and at least one non-target analyte or material is stained.

[0053] After staining, the sample can be imaged, whereby the stained sample is illuminated with excitation light of one or more wavelengths from a light source, such as a laser or a light emitting diode, such as infrared, red, blue, green, and / or ultraviolet light. Imaging can be accomplished using a flow cytometer or a microscope, such as a fluorescence microscope, a scanner, or any other appropriate imaging system or means. In one embodiment, imaging can be performed in a system that detects groups that can provide signals across the optical spectrum when imaged, including but not limited to brightfield and / or darkfield illumination, fluorescence, and the like. When multiple detection groups are used, the images formed can be overlaid. Emission, reflection, diffraction, scattering, and combinations thereof are used for detection / imaging. The images can be analyzed to detect, enumerate, and / or locate target analytes, such as when it is desired to retrieve or pick target analytes. Imaging is performed in tubes, on microscope slides, or in any suitable container or substrate for imaging.

[0054] The methods can be performed 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 methods can be performed by any combination of the above. The methods described can be used in a system that detects groups that can provide signals across the optical spectrum when imaged, including but not limited to brightfield and / or darkfield illumination, fluorescence, and the like.

[0055] Imaging chain for an imaging system

[0056] In a fluorescence microscope, fluorophores (or fluorescent dyes) are used to stain samples, such as proteins or other molecules, tissues, and cells of interest, for examination or study. A fluorophore 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 fluorophore has a specific absorption or excitation wavelength band, and the excitation filter is chosen to transmit the excitation wavelength range. Once excited, the fluorophore emits a range of wavelengths. The emission filter transmits the desired emission wavelength. The dichroic filter, which is specifically 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 can also be designed to reflect the emission wavelength and transmit the excitation wavelength.

[0057] Figure 1One embodiment of an imaging chain of a fluorescence microscope imaging system is shown. The optical path can include an excitation source 102 that emits at least one excitation light 104, such as light in the visible, infrared ("IR"), or ultraviolet ("UV") spectrum. The excitation source 102 can include a laser light source, an LED light source, a xenon light source, a halogen light source, an incandescent light source, or other appropriate light source. In some embodiments, the excitation light 104 includes multiple wavelengths, including at least a first excitation wavelength 106 and a second excitation wavelength 108. In such embodiments, the excitation light 104 can interact with an excitation spectral selector 110 such that the first excitation wavelength 106 passes through the excitation spectral selector 110 and the second excitation wavelength 108 is blocked from passing through the excitation spectral selector 110. In particular embodiments, the excitation spectral selector 110 can include one or more optical or interference filters, such as one or more variable excitation filters. In one such embodiment, after the first excitation wavelength 106 exits the excitation filter of the excitation spectral selector 110, it is then reflected off of a second filter 112. The second filter 112 redirects the first excitation wavelength 106 into an objective lens 114. The second filter 112 can be dichroic, multichroic, short-pass, long-pass, band-pass, band-reject, or any appropriate filter.

[0058] With continued reference to Figure 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 surface on, in, or near the sample or portion 134. The first excitation wavelength 106 stimulates a first detection moiety (not shown) on or in the sample or portion 134, thereby enabling the first detection moiety (not shown) to emit a first emission wavelength signal 116. The first emission wavelength signal 116 can be captured by the objective lens 114, transmitted through the second filter 112 configured to transmit the first emission wavelength, then transmitted through an emission spectral selector 130 onto an emission detector 140, where an original image is captured or acquired. The emission detector 140 can be a charge-coupled device ("CCD"), a CMOS camera, a scientific CMOS camera, a photodiode, a photomultiplier tube, or the like, for capturing image data, which can then be compiled into an image, processed, and analyzed by a computer or associated software or program. In certain embodiments, the second filter 112 is a dichroic filter configured to reflect the shorter wavelengths that typically excite a fluorophore and transmit the longer wavelengths emitted by the fluorophore. The second filter 112 and the emission spectral selector 130 together are configured to limit or prevent non-emission energy and stray light from reaching the sensor of the emission detector 140.

[0059] In other embodiments, the excitation source 102 emits the excitation light 104, which then interacts with the excitation spectral selector 110 such that the second excitation wavelength 108 passes through the excitation spectral selector 110 and the first excitation wavelength 106 is blocked from passing through the excitation spectral selector 110. The second excitation wavelength 108 is then reflected off the second filter 112, thereby redirecting 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 at a point or surface on, in, or near the sample or portion thereof 134. The second excitation wavelength 108 stimulates a second detection moiety (not shown) on or in the sample or portion thereof 134, thereby enabling the second detection moiety (not shown) to emit a second emission wavelength signal 118. The second emission wavelength signal 118 can be captured by the objective lens 114, transmitted through the second filter 112, pass through the emission spectral selector 130 onto the emission detector 140, where an original image can be captured. The described process can be performed one or more times for a desired number of detection moieties.

[0060] In alternative embodiments, the excitation source 102 can be configured to emit the excitation light 104 as one or more individual wavelengths, such as the first excitation wavelength 106 and the second excitation wavelength 108. In other embodiments, Figure 1 The optical path of the fluorescence microscope shown in FIG. 1 can optionally not include the excitation spectral selector 110.

[0061] With continued reference to Figure 1 The sample or portion thereof 134 can be located on a base 132 or between a cover 136 and the base 132. The cover 136 and the base 132 can be optically clear or optically transparent to permit imaging. In some embodiments, the base 132 and the cover 136 can be constructed from one or more of the following: glass; inert metals; metals; metalloids; organic or inorganic materials and plastic materials such as polymers; and combinations thereof.

[0062] The sample 134, the cover 136, and the base 132 can be located on a stage 128 to move the sample 134 in x, y, or z directions as desired. The stage 128 can include an aperture 138 that enables the first excitation wavelength 106 that has been focused by the objective lens 114 to pass into, on, or near the sample or portion thereof 134. The stage 128 can be driven by a driver 120 that includes at least one of a z-direction driver 124, an x-direction driver 122, and a y-direction driver 126 to position the sample 134. The driver 120 can be a motor such as a servo motor or a stepper motor, a piezoelectric actuator, a solenoid, or the like.

[0063] The optical path may also include, for example, a cut-off aperture (not shown) in a confocal microscope, to increase the signal-to-noise ratio of the boundary light signal.

[0064] 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 allow light of a desired wavelength to pass through. In one embodiment, the excitation spectrum selector 110 or the emission spectrum selector 130 may be a notch filter, a bandstop filter, a long-pass filter, a short-pass filter, a bandpass filter, or a multicolor filter. In one embodiment, the excitation spectrum selector 110 or the emission spectrum selector 130 may be a diffraction grating. In another embodiment, the excitation spectrum selector 110 or the emission spectrum selector 130 may include a variable-angle or variable-tilt filter capable of re-angled to block or allow selected wavelengths to pass through by changing the relative angle of incidence of the incoming excitation or emission light on the filter. As an example, a first excitation wavelength 106 passes through the excitation spectrum selector 110, and a 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. Alternatively, the excitation spectrum selector 110 can be selectively angled to block the first excitation wavelength 106 and allow the second excitation wavelength 108 to pass through. As used herein, the angle of incidence is the angle (θ) between the light ray incident on the surface at the point of incidence and a line perpendicular to the surface.

[0065] In another embodiment, the first emission wavelength 116 passes through the emission spectrum selector 130, and the second emission wavelength signal 118 is blocked from passing through the emission spectrum selector 130 at least in part due to the angle of the emission spectrum selector 130. Alternatively, the emission spectrum selector 130 can be selectively angled to block the first emission wavelength signal 116 and allow the second emission wavelength signal 118 to pass through.

[0066] Continue to refer to Figure 1 The emission spectrum selector 130 may include at least one or more interference filters, or more specifically, 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 such that each is a variable filter capable of tilting or angled on axes parallel to or alternatively perpendicular to each other. The first emission filter 142 may be tilted or angled to achieve a desired angle of incidence between the first emission filter 142 and the emitted light (represented by the longer dashed line filter 142). Similarly, the second emission filter 144 may be tilted or angled to achieve a desired angle of incidence between the second emission filter 144 and the emitted light (represented by the longer dashed line filter 144).

[0067] The first emission filter 142 and the second emission filter 144 can be tilted or angled independently of one another. In certain embodiments, the first emission filter 142 can be variably positioned to any desired position (i.e., a first position, a second position, a third position, a fourth position, and so on, up to an n position), where each position corresponds to a different angle Θ. Likewise, the second emission filter 144 can be variably positioned to any desired position (i.e., a first position, a second position, a third position, a fourth position, and so on, up to an n position), where each position corresponds to a different angle Θ. The first emission filter 142 and the second emission filter 144 can have independent positions or angles from one another, such that one or both of the first emission filter 142 and the second emission filter 144 can be angled or tilted to the same angle of incidence or to different angles of incidence with respect to one or more emission rays. In particular embodiments, the first emission filter 142 and the second emission filter 144 can be angled to pass or block emission light of a desired wavelength.

[0068] For example, a first raw image can be obtained with the first emission filter 142 at a first angle of incidence and with the second emission filter 144 at a third angle of incidence. Then, the first emission filter 142 can be re-angled from the first angle of incidence to a second angle of incidence, while the second emission filter 144 remains at the third angle of incidence. Then, a second raw image can be obtained. Additionally, while capturing the at least one or more raw images, the second emission filter 144 can be re-angled from the third angle of incidence to a fourth angle of incidence. Then, a third raw image can be obtained. In one embodiment, at least two of the first angle of incidence, the second angle of incidence, the third angle of incidence, and the fourth angle of incidence are the same. In one embodiment, none of the first angle of incidence, the second angle of incidence, the third angle of incidence, and the fourth angle of incidence are the same.

[0069] In any of the embodiments that include tilting or angling at least one filter, any filter can be tilted or angled at any desired time or in any desired order to block or pass a desired emission wavelength range. For example, after obtaining a first raw image with the first emission filter 142 at a first angle of incidence, the first emission filter 142 can be tilted or angled to a second angle of incidence. Then, a second raw image can be obtained. Then, the first emission filter 142 can be tilted or angled again, and then a third raw image can be obtained. In other words, each filter can be tilted or angled at any amount at any point independent of the other filter or filters to obtain any raw image and / or any desired range of emission wavelength ranges.

[0070] Further, any number of filters can be used, including but 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.

[0071] Some particular examples and embodiments of excitation spectral selector 110 can include one or more variable interference filters configured such that the wavelength blocking and transmission can be tuned by tilting the filter and changing the angle of incidence of the light path on the filter. In some embodiments, at least one of excitation spectral selector 110 and / or emission spectral selector 130 can include at least one variable interference filter that can be tilted or angled. In certain such embodiments, both excitation spectral selector 110 and emission spectral selector 130 can include at least one variable interference filter. In such embodiments, the variable interference filter can be an excitation filter, an emission filter, a polychromatic filter, and configured for use with a multi-channel fluorescence microscope and high-throughput imaging system.

[0072] The individual filters of excitation spectral selector 110 or emission spectral selector 130 and / or the angle of incidence between the filters and the excitation or emission light rays can be selected to provide the desired wavelengths for capturing raw images at a selected band of the emission spectrum of the one or more detection groups. For example, the interference filters included in excitation spectral selector 110 and / or emission spectral selector 130 are configured to block and / or transmit the desired wavelengths along the light path to capture raw images at locations on the lower and upper edges of the spectral edges of the one or more emission spectra. For example, in one embodiment, the detection groups can have spectral differences at their peak values that are less than or equal to 50 nm. In one embodiment, the detection groups can have differences in their peak values in the spectrum that are less than or equal to 10 nm. In one embodiment, the detection groups have differences in their peak values in the spectrum that are 1-50 nm. In one embodiment, the detection groups can be separated by a few nanometers at their peak values, including but 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 nm. In one embodiment, the difference between consecutive spectra (such as at peak values) can be the same (e.g., a first detection group and a second detection group are separated by 10 nm, and a second detection group and a third detection group are separated by 10 nm). In one embodiment, the difference between consecutive spectra (such as at peak values) can be different (e.g., a first detection group and a second detection group are separated by 10 nm, and a second detection group and a third detection group are separated by 25 nm).

[0073] In one embodiment, the angle of incidence Θ of light on any optical filter can be any angle, including but not limited to 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 approximate angle of incidence of light on any optical filter can be up to 90° but not including 90°. In one embodiment, the approximate angle of incidence of light on any optical filter can be less than 90°. In one embodiment, the approximate angle of incidence of light on any optical filter can be from 0.0° to 89.9°. In certain embodiments, when there are two or more tiltable optical filters, each optical filter can be free to tilt independently of the other optical filters, such that two or more optical filters can have the same angle of incidence, or no two optical filters have the same angle of incidence. In particular embodiments, the angle of incidence is selected based on the desired wavelengths to be passed or blocked under the action of the optical filter.

[0074] Filter translator

[0075] The imaging chain of the imaging systems disclosed herein can include filter changers configured to change quickly and easily between multiple optical filters. In certain embodiments, the filter changer can be a filter slider or filter wheel configured to hold one or more optical filters. The filter changers disclosed herein can be configured for use in systems and methods of multi-channel fluorescence microscopes and automated high-throughput imaging systems. In some embodiments, at least one of the excitation spectral selector 110 and / or the emission spectral selector 130 can be configured to include at least one filter changer capable of tilting or angling one or more variable optical filters, such as one or more variable interference filters. For example, with reference to Figure 1 , one or both of the first or second emission filters 142, 144 can be a variable interference filter held by a filter changer having one or more variable interference filters.

[0076] With reference to Figure 2AOne embodiment of a filter changer can be a rotating filter wheel, the filter wheel 200 includes one or more optical filters, such as variable interference filter 242, that can be disposed in the optical path of the imaging chain. The filter wheel 200 can be configured to rotate on or about an axis or shaft at the hub 250 in a clockwise or counterclockwise direction. The filter wheel 200 can be designed to have a position indicator or detent at a desired location of the filter wheel 200. For example, the filter wheel 200 can be manually rotated or rotated by a motor having a shaft attached to the hub 250 in order to dispose the variable interference filter 242 at any desired filter position, such as filter position 252. In a preferred embodiment, the filter position 252 is in the optical path of the light beam traveling through the imaging chain, thereby positioning the variable interference filter 242 in the optical path of the light beam. For example, the filter wheel 200 is rotated by a motor having a shaft attached to the hub 250 to dispose the variable interference filter 242 in the optical path of one or both of the first emission wavelength signal 116 and the second emission wavelength signal 118 (see also Figure 1 and Figure 4B ).

[0077] In some embodiments, a filter changer, such as a filter slider or filter wheel 200, can include at least one filter assembly configured to hold an optical filter. As shown in Figure 2A , the filter wheel 200 can include at least one filter assembly 260 configured to hold an optical filter, such as variable interference filter 242. In certain embodiments, the filter wheel 200 can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and more filter assemblies 260. In one embodiment, the filter wheel 200 includes two or more of the filter assemblies 260 and each filter assembly 260 includes an optical filter, wherein no two optical filters have all the same optical filter properties. In another embodiment, each of the two or more of the filter assemblies 260 includes an optical filter, wherein at least two filters have the same filter properties. In other embodiments, the filter changer can include at least one filter assembly 260 that does not hold a filter (such as a filter assembly 260 that includes an optical window, clear glass), or the filter assembly 260 can not hold anything.

[0078] Referring to Figure 2BSome embodiments of the filter assembly 260 are configured to hold an interference filter, such as the variable interference filter 242, within a housing having a base 264 and a holder 266. In one embodiment, the base 264 and the holder 266 can include one or more compression clips 276 and a window 274 configured to permit light to pass through the variable interference filter 242. In particular embodiments, the base 264 can also include at least one bearing 278 configured to tilt, rotate, and / or translate the filter assembly 260 when attached to the filter wheel 200. In one embodiment, the at least one bearing 278 can be configured to enable the filter assembly 260, including the variable interference filter 242, the base 264, and the holder 266, to tilt and / or translate when attached to the filter wheel 200 to change the angle of incidence between the variable interference filter 242 and an excitation or emission light ray in the optical path of the imaging chain.

[0079] Referring now to Figure 3 In certain embodiments of an imaging chain for an imaging system including the filter wheel 200, the tilt mechanism can be configured to engage with and tilt a filter assembly. For example, a tilt mechanism, such as the cam 300, can be configured to engage with the filter assembly 260 when the filter assembly 260 is positioned in the optical path. In particular embodiments, the tilt mechanism, such as the cam 300, is configured to engage with 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 an emission or excitation light ray in the optical path.

[0080] The cam 300 can include a body 302 and a tail 304 extending from the body 302, the tail including an engagement surface 308 for engaging the bearing 272 of the filter assembly 260. The tail 304 of the cam 300 can be any suitable shape or configuration, for example, rectangular, cuboid, triangular, pyramidal, curved, hooked, horn-shaped, or combinations thereof, among others. Further, the size and shape of the tail 304 can be determined to avoid bumping into any other components as the cam is rotated. In one embodiment of the cam 300, the body 302 includes a hole 310 extending at least partially through the body 302 to mate with the motor 320 or a connector to abut the motor 320 and the cam 300. In one embodiment, the body 302 includes a shaft or other connector to mate with the motor 320. In another embodiment, the cam 300 and the motor 320 can be an integrated unit or a single piece.

[0081] In one embodiment of filter wheel 200, filter assembly 260 and variable interference filter 242 at filter position 252 can be tilted as a result of engagement of bearing 272 with cam 300 such that the angle of incidence of light on the filter can be any angle of incidence ranging from approximately 0.0° to 89.9°. For example, the angle of incidence of light on the desired filter can be approximately, but not limited to, 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 certain embodiments, when there are two or more variable interference filters positioned in the optical path, each filter can be configured to be free to tilt independently of the other filters such that two or more filters can have the same angle of incidence or no two filters have the same angle of incidence. The angle of incidence can be selected based on the desired wavelengths to be blocked or passed under the action of the filter.

[0082] Correction of optical aberrations

[0083] As light rays travel through the imaging train of a fluorescence microscope described herein, they encounter optical elements such as optical windows and optical filters made of glass and other substrates. Light rays that are incident on the glass surface of a filter will be distorted as they are refracted and enter and exit from air into glass. Refraction and distortion of light by optical elements introduce optical aberrations that can cause a decrease in optical system performance and image quality. Optical aberrations can include astigmatism, lateral image shift, focal shift, spherical aberration, chromatic aberration, coma, vignetting, and variable spectral selection across the field of view. Optical aberrations can cause image registration errors, focusing errors, and loss of image resolution and accuracy. Introducing optical aberrations is particularly problematic when using a multi-channel fluorescence microscope system that relies on the alignment of multiple images captured from various detection moieties, different fluorophores, and filter sets. Embodiments of imaging trains of fluorescence microscope imaging systems for minimizing, limiting, eliminating, and / or correcting optical aberrations are described herein.

[0084] Reference Figure 4A and Figure 4BEmbodiments of an imaging train for a fluorescence microscope system can include a tiltable or variable optical filter, such as a variable interference filter 242 clamped by filter changer 400 and disposed in the optical path. In some embodiments, the variable optical filter can be used to select an emission band spectrum or emission wavelength as it generally provides significantly better transmission efficiency compared to alternative methods such as liquid crystal based variable filters. Excitation or emission light rays are refracted and distorted as they pass through the variable interference filter 242, often resulting in lateral image shifts and astigmatism. Any resulting lateral image shifts and astigmatism change as the angle of incidence of the variable interference filter 242 changes. As disclosed herein, optical elements can be placed in the optical path to mitigate, minimize, or correct for the changing optical aberrations caused by the variable interference filter 242. For example, in certain embodiments, a dynamic correction optic 410 can be configured to work with the variable interference filter 242 to produce a predictable and substantially constant lateral image shift and astigmatism for any desired angle of incidence of the variable interference filter 242. In other embodiments, a fixed correction optic 420 can be disposed in the optical path and configured to substantially mitigate or minimize any astigmatism caused by the variable interference filter 242 and the dynamic correction optic 410. In other embodiments, the fixed correction optic 420, the dynamic correction optic 410, and the variable interference filter 242 can be disposed in the optical path and configured to simultaneously minimize astigmatism, lateral image shift, and focus shift.

[0085] Referring to Figure 4B Embodiments of the imaging train can include a dynamic correction optic 410 that is tiltable in the X-axis 412, which is substantially perpendicular to the Y-axis 422 over which the fixed correction optic 420 has been tilted. The dynamic correction optic 410 can be a flat optically transparent plate, such as an optical window formed from a desired substrate. The dynamic correction optic 410 can be positioned in the optical path of the imaging train either before or after the variable interference filter 242.

[0086] In one embodiment, the dynamic correction optic 410 is supported by a dynamic optical assembly 414 having a hub 416. In certain embodiments, the hub 416 is positioned substantially along the X-axis 412. The dynamic correction optic 410 can be selectively tilted about the X-axis 412 manually or by a motor and / or the engagement of a shaft with the hub 416. In particular embodiments, the angle of incidence of light in the optical path on the surface of the dynamic correction optic 410 can be any angle of incidence in the range of approximately 0.0° to 89.9°. For example, the angle of incidence of light on the dynamic correction optic 410 can be approximately, but not limited to, 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°.

[0087] Referring to Figure 4A and Figure 4B The dynamic correction optic 420 can be a flat optically transparent plate, such as an optical window formed from a desired substrate. The fixed correction optic 420 is placed in the optical path of the imaging train at a fixed angle of tilt about the Y-axis 422 selected from any angle in the range of approximately, but not limited to, 0° to 90°, 10° to 80°, 20° to 70°, 30° to 60°, and 40° to 50° relative to the optical path of the imaging train. In particular embodiments, the fixed angle of the fixed correction optic 420 is any angle that can substantially compensate for one or more of the optical aberrations that can be introduced by one or more optical elements in the imaging train.

[0088] As described herein, the fixed correction optic 420 is tilted on the Y-axis 422 and the dynamic correction optic 410 is tilted on the X-axis 412. Referring to Figure 4B The variable interference filter 242 is tilted on an X-axis that is approximately parallel to the X-axis 412 and approximately perpendicular to the Y-axis 422. As such, the variable interference filter 242 and the dynamic correction optic 410 are configured to be tilted on approximately parallel X-axes that are approximately perpendicular to the Y-axis 422 about which the fixed correction optic 420 is tilted.

[0089] The dynamic correction optic 410 and the variable interference filter 242 can be tilted independently of one another. In certain embodiments, the variable interference filter 242 can be variably tilted to any desired position (i.e., a first position, a second position, a third position, a fourth position, and so on, up to an n-th position) that passes or blocks the emission light of a desired wavelength, where each position corresponds to a different angle. Likewise, the dynamic correction optic 410 can be variably tilted to any desired position (i.e., a first position, a second position, a third position, a fourth position, and so on, up to an n-th position), where each position corresponds to a different angle. The dynamic correction optic 410 and the variable interference filter 242 can have independent positions or angles from one another, such that one or both can be tilted to the same angle of incidence or different angles of incidence with respect to one or more emission rays.

[0090] For example, during the capture of a plurality of raw images, a first raw image can be obtained with the variable interference filter 242 at a first angle of incidence and with the dynamic correction optic 410 at a corresponding third angle of incidence. For a second raw image, the variable interference filter 242 can be re-angled from the first angle of incidence to a second angle of incidence, while the dynamic correction optic 410 is tilted to a corresponding fourth angle of incidence. In this manner, the dynamic correction optic 410 can be configured to work with the variable interference filter 242 to produce a predictable and approximately constant lateral image shift and astigmatism for any desired angle of incidence of the variable interference filter 242.

[0091] The variable interference filter 242, the dynamic correction optic 410, and the fixed correction optic 420 need not be positioned in a particular order in the imaging chain. In some embodiments, the optical path of the imaging chain passes through the fixed correction optic 420, the dynamic correction optic 410, and the filter wheel 200 in that order.

[0092] A. Astigmatism

[0093] During operation of the fluorescence microscope, the imaging chain as described herein is configured to substantially mitigate, minimize, remove, and / or eliminate optical aberrations produced by one or more of the included optical elements. With continued reference to Figure 4A and 4B In one embodiment, the fixed correction optic 420 is configured to substantially mitigate and / or approximately eliminate astigmatism introduced by both the dynamic correction optic 410 and the variable interference filter 242. In another embodiment, the residual astigmatism caused or introduced by the combination of the fixed correction optic 420, the dynamic correction optic 410, and the variable interference filter 242 is approximately zero (0).

[0094] In such embodiments, the combined astigmatism induced by the dynamic correction optics 410 and the variable interference filter 242 is configured to be approximately constant, even if the variable interference filter 242 is selectively tilted about the X-axis. As such, for a selected angle of the variable interference filter 242, the dynamic correction optics 410 are also tilted such that their combined astigmatism approximately cancels the astigmatism induced by the fixed correction optics 420.

[0095] B. Lateral Image Shift

[0096] In another embodiment, the sum of the lateral image shift introduced by both the dynamic correction optics 410 and the variable interference filter 242 remains approximately constant for any desired angle of incidence of the variable interference filter 242. For example, the combined lateral shift induced by the dynamic correction optics 410 (LSHFT 410 ) and the variable interference filter 242 (LSHFT 242 ) is approximately constant (LSHFT Constant ). In another form:

[0097] LSHFT Constant = LSHFT 410 + LSHFT 242 .

[0098] Thus, for a selected angle of incidence of the variable interference filter 242, the combined lateral image shift induced by the dynamic correction optics 410 (LSHFT 410 ) and the variable interference filter 242 (LSHFT 242 ) is an approximately constant value, even if the variable interference filter 242 is selectively tilted about the X-axis.

[0099] C. Focus Shift

[0100] In other embodiments, the imaging chain described herein is configured to substantially stabilize the total focus shift induced by the refraction through the dynamic correction optics 410 and the variable interference filter 242. When the focus shift is stabilized, the system focus can be shifted by adjusting the sensor or camera and / or the objective along the optical axis to achieve the desired image focus.

[0101] Telecentric tube lens

[0102] An infinity-corrected objective is a microscope objective that is focused at infinity. To create an image with an infinity-corrected objective, an image can be focused at the image plane using a tube lens. One advantage of using an infinity-corrected objective with a tube lens is that there can be space between the objective and the tube lens that allows additional optical components, such as optical filters, to be inserted into the system. In some embodiments described herein, a telecentric tube lens configured for use with an infinity-corrected objective can be disposed in the optical path of a fluorescence microscope to minimize, mitigate, limit, eliminate, or correct for optical aberrations such as vignetting, chromatic aberration, spherical aberration, and variable spectral selection across the field of view. The use of a telecentric tube lens can also minimize any need for digital compensation of optical aberrations, thereby enabling image processing to run faster by reducing CPU load, which results in higher system throughput.

[0103] Reference Figure 5 Embodiments of an imaging chain for a fluorescence microscope system can include a dual telecentric tube lens 500. A telecentric tube lens is a compound lens with an entrance pupil or an exit pupil at infinity. A telecentric tube lens produces chief rays that are parallel to the optical axis of the optical path. Parallel chief rays are desirable because they produce a selected spectrum that is uniform across the field of view when they pass through a tilted or variable optical filter, such as a variable interference filter 242 Figure 4B

[0104] In certain embodiments, the dual telecentric tube lens 500 is configured to be positioned at a location 510 upstream of a tilted or variable interference filter, such as a variable interference filter 242. The location 510 can be in the optical path of the dual telecentric tube lens 500 that is telecentric in both image space and object space. In such embodiments, the dual 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 dual telecentric tube lens 500 is configured to produce chief rays that are substantially parallel and have a substantially zero (0) angle of incidence at the emission detector 140. Because the chief rays are parallel, variable spectral selection, spherical aberration, chromatic aberration, coma, and vignetting across the field of view can be minimized or limited, thereby improving image resolution and quality.

[0105] Low incidence filter

[0106] ​Generally, dichroic and multichroic filters are configured to have desired passbands and stopbands designed to operate at standard 45° angles of incidence. However, lower angles of incidence enable better filtering performance, including improved blocking, transmission, steeper bandpass edges, and beam collimation. Embodiments of an imaging train for a fluorescence microscope are disclosed herein that include a multichromatic excitation filter having a lower or narrower angle of incidence of excitation light than the standard 45° angle of incidence.

[0107] Reference is made to Figure 6 The optical path can include an excitation source 102 that emits excitation light 104 directed toward an excitation spectral selector 110 that 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 is transmitted by the excitation filter of the excitation spectral selector 110, it is then directed toward a low incidence filter 600 that is positioned to form a low angle of incidence with the optical path. In certain embodiments, the angle of incidence of the low incidence filter 600 can be any angle of incidence ranging from approximately 10.0° to 30.0°. For example, the angle of incidence of the light on the low incidence filter 600 can be approximately, but not limited to, 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°.

[0108] At the selected angle of incidence Θ, the low incidence filter 600 is configured to reflect the first excitation wavelength 106 and / or the second excitation wavelength 108 into the objective lens 114 and enable transmission of the first emission wavelength signal 116 and / or the second emission wavelength signal 118. The narrow incidence filter 600 can be a dichroic, multichroic, shortpass, longpass, bandpass, bandstop, multiband, or any desired filter.

[0109] Spectral edge detection

[0110] When multiple detection moieties or fluorophores are used in multi-channel, polychromatic fluorescence microscopy, spectral overlap or crosstalk can limit the ability to distinguish individual detection moiety signals. As disclosed herein, spectral edge detection is one such process by which individual detection moieties can be distinguished from multiple detection moieties (i.e., during multiplexing) such as by orthogonally distinguishing detection moieties - because there is no ambiguity as to which detection moiety is being detected and / or imaged. Improvements to spectral edge detection performance can be achieved by minimizing, limiting, and / or eliminating optical aberrations in the imaging chain for multi-channel, polychromatic fluorescence microscopy.

[0111] For some embodiments of the fluorescence microscopy described herein, raw images are acquired by an emission detector at selected wavelengths of an emission spectrum. Each raw image can include a total selected emission spectrum signal. Each total signal can include one or more signals from one or more detection moieties. Each total signal can also include a signal due to background or autofluorescence. For spectral edge detection, a signature of a signal of interest (such as a signal from a detection moiety of interest) can be distinguished in the presence of a non-signature signal such as from background, autofluorescence, or an undesired detection moiety (i.e., a signal with an unknown value and / or structure). In other words, when the intensity (and thus the corresponding contribution) of a detection moiety is unknown, spectral edge detection determines the contribution of a detection moiety of interest on a plurality of signals (or images) composed of contributions from multiple detection moieties with at least partially overlapping spectra by eliminating the contribution from undesired detection moieties on the plurality of signals (or images).

[0112] Spectral edge detection can also account for minor variations in signals, for example, when detecting a detection moiety against a reference detection moiety, or when incorporating a detection moiety with an emission shift based on one or more factors, whether intentional (e.g., a detection moiety that includes a sample variable such as oxygen concentration, metal ion concentration, environmental changes, endocytosis, exocytosis, etc.) or unintentional (e.g., a variable pH of a sample or reagent causes a detection moiety to have an emission shift).

[0113] Spectral edge detection uses an edge (e.g., a trailing edge or a leading edge) of an emission or excitation spectral curve, such as an emission or excitation spectral curve for a detection moiety, to identify an individual detection moiety within a plurality of spectrally overlapping detection moieties. For example, two raw images can be acquired along the same spectral leading or trailing edge of a detection moiety emission spectral curve; two raw images can be acquired along different spectral leading and trailing edges of a detection moiety emission spectral curve; or one raw image can be acquired along a spectral leading or trailing edge of a detection moiety emission spectral curve, and one raw image can be acquired at a peak emission of a detection moiety emission spectral curve.

[0114] Spectral edge detection can also use a combination of the examples and methods discussed herein for a single detection group or for multiple detection groups. For example, when using multiple detection groups, a first detection group can be detected with signals from the peak and the spectral leading edge; a second detection group can be detected with signals from the spectral leading edge; a third detection group can be detected with signals from the spectral leading edge and the spectral trailing edge. Further, spectral edge detection can utilize a curve for the first detection group and a straight line for the second detection group, such that at least a portion of the straight line falls below the curve with data points having each emission spectrum (e.g., signals at a given emission / excitation wavelength).

[0115] Figure 7A An emission spectrum 702 for a first detection group is shown. The emission spectrum 702 includes a spectral leading edge 704 and a spectral trailing edge 706. In other words, the spectral leading edge 704 is a portion of the emission spectrum 702 to the left of or having a shorter wavelength than a peak emission 708; the spectral trailing edge 706 is a portion of the emission spectrum 702 to the right of or having a longer wavelength than the peak emission 708. Although an emission spectrum 702 is shown, the spectrum can also be an excitation spectrum.

[0116] Figure 7B An emission spectrum 710 for a second detection group is shown. The emission spectrum 710 includes a spectral leading edge 712 and a spectral trailing edge 714. In other words, the spectral leading edge 712 is a portion of the emission spectrum 710 to the left of or having a shorter wavelength than a peak emission 716; the spectral trailing edge 714 is a portion of the emission spectrum 710 to the right of or having a longer wavelength than the peak emission 716. Although an emission spectrum 710 is shown, the spectrum can also be an excitation spectrum.

[0117] Figure 7C An emission spectrum 720 for a third detection group is shown. The emission spectrum 720 includes a spectral leading edge 722 and a spectral trailing edge 724. In other words, the spectral leading edge 722 is a portion of the emission spectrum 720 to the left of or having a shorter wavelength than a peak emission 726; the spectral trailing edge 724 is a portion of the emission spectrum 720 to the right of or having a longer wavelength than the peak emission 726. Although an emission spectrum 720 is shown, the spectrum can also be an excitation spectrum.

[0118] In one embodiment, any of the methods or systems can be used to detect a stain or detection moiety when removing background or autofluorescence from an image or signal. For example, two or more raw images of a first detection moiety are provided such that at least one of the images is at a lower end of a spectral edge of the first detection moiety and at least one of the images is at a higher end of the spectral edge of the first detection moiety. At least one of the raw images includes a signal caused by autofluorescence or background. A first final image of the first detection moiety is provided such that the first final image is based on the raw images of the first detection moiety and the first final image does not include the signal caused by autofluorescence or background. This can be performed for any number of detection moieties to remove background or autofluorescence from any image.

[0119] In Figure 8A and Figure 8B , the fourth detection moiety (as depicted by emission spectrum 804) is used as an example of a method for distinguishing the individual detection moieties from background or autofluorescence. However, it should be noted that the methods discussed herein are not limited to this and can also be implemented on the first, second, and / or third detection moieties (as depicted by emission spectra 702, 710, 720) or any other appropriate detection moieties.

[0120] Figure 8A The emission spectrum 804 and the background signal 802 are shown. The background signal 802 is expected to be relatively constant with respect to the signal of interest, and thus is depicted as a constant (i.e., a straight line) with a known value. Additionally, the relative intensities between the emission spectrum 804 and the background signal 802 are unknown.

[0121] For clarity, Figures 8B to 9E Images I1-I 18 are depicted from the indicated single wavelengths. However, images I1-I 18The images (as represented by the double-dotted lines) represent the average signal over the respective bandwidths can be obtained over a given bandwidth (i.e., up 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, 100 nm, 150 nm, 200 nm, or more nm) such that the images (as represented by the double-dotted lines) represent the average signal over the respective bandwidths. Moreover, while the original images are obtained, the signals (e.g., including the individual signals) are from pixels in the original images that correspond to the same location of the sample in the individual images. For example, point A is a given location on or within the sample being imaged. A first pixel in a first original image representing point A includes a first total signal. A second pixel in a second original image representing point A includes a second total signal. The first and second total signals of the first and second pixels of the first and second original images are evaluated and / or compared, respectively, using one or more methods discussed herein to determine the contribution(s) of the individual detection moieties.

[0122] Figure 8B Original images Ii and I2 are shown that include first and second signals Si and S2, respectively, obtained during imaging. The signals Si and S2 represent the total contribution of the fourth detection moiety and background 802. Original image Ii includes first signal Si on the lower end of the spectral leading edge; and original image I2 is taken at the higher end of the spectral leading edge. To identify the fourth detection moiety (as shown by emission spectrum 804), the original images Ii and I2 are analyzed and the relative contribution of the fourth detection moiety between the first and second signals Si and S2 is determined by processing, comparing, and / or analyzing the changes in the signals with any appropriate mathematical, computational, or algebraic process or transformation, including but not limited to subtraction, derivative, or combinations thereof. A final image depicting the fourth detection moiety can then be provided based on the processing, comparing, and / or analyzing.

[0123] Spectral edge detection can be implemented for each detection moiety within a plurality of detection moieties, thereby enabling a sample or portion thereof to be multiplexed with any desired number of detection moieties. In one embodiment, at least two detection moieties can be used for multiplexing. In one embodiment, any appropriate number of detection moieties can be used, including but not limited to 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.

[0124] Spectral edge detection can be implemented for detection groups with spectral offsets, where a spectral offset is a spectral difference at a comparable spectral edge or spectral peak. In one embodiment, the process can be implemented for detection groups with spectral offset differences less than or equal to 50 nm. In one embodiment, the process can be implemented for detection groups with spectral offset differences less than or equal to 10 nm. In one embodiment, the process can be implemented for detection groups with spectral offset differences of 1 nm to 50 nm. In one embodiment, the process can be implemented for detection groups with spectral differences of 10 nm to 50 nm. In one embodiment, the process can be implemented for detection groups with spectral offset differences of approximately, but 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 nm. In one embodiment, the difference between consecutive spectra, such as at a peak, can be the same (e.g., the first detection group and the second detection group are separated by 10 nm, and the second detection group and the third detection group are separated by 10 nm). In one embodiment, the difference between consecutive spectra, such as at a peak, can be different (e.g., the first detection group and the second detection group are separated by 10 nm, and the second detection group and the third detection group are separated by 25 nm).

[0125] In one embodiment, the signal contribution of each detection group (e.g., by a contribution or subtraction coefficient) can be determined with at least two raw images, such as by canceling or eliminating the signal contribution provided by a non-focus detection group (i.e., the first detection group is a non-focus detection group and the second detection group is a focus detection group; and / or, the first detection group is a focus detection group and the second detection group is a non-focus detection group), or background / spontaneous fluorescence. Any number of raw images equal to or greater than 2 can be obtained for spectral edge detection, including but 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.

[0126] For clarity, with respect to Figures 9A to 9E , the emission spectrum 702 of the first detection group is also denoted by “A”; the emission spectrum 710 of the second detection group is also denoted by “B”; and, the emission spectrum 720 of the third detection group is also denoted by “C”. In this specification, a subscript after A, B, or C denotes the image for which the focus detection group contributes intensity. For example, data point A3 denotes the contribution of A (or the first detection group) within raw image I3, as shown by data point A3 on the emission spectrum 702. Thus, A3– A 18respectively represent the contribution of A (or the first detection moiety) in the original images I3-I 18 respectively represent the contribution of B (or the second detection moiety) in the original images I3-I 18 respectively represent the contribution of B (or the second detection moiety) in the original images I3-I 18 respectively represent the contribution of B (or the second detection moiety) in the original images I3-I 18 respectively represent the contribution of C (or the third detection moiety) in the original images I3-I 18 respectively represent the contribution of C (or the third detection moiety) in the original images I3-I

[0127] Figure 9A Emission spectrum 702 for the first detection moiety and emission spectrum 710 for the second detection moiety are shown. Original images I3, I4, and I5 are obtained at the wavelength positions indicated on emission spectrum 702 and emission spectrum 710. Original image I3 is taken at the lower end of the spectral leading edge 704 of emission spectrum 702; original image I4 is taken at the higher end of the spectral leading edge 704 of emission spectrum 702 that also overlaps the lower end of the spectral leading edge 712 of emission spectrum 710; and original image I5 is taken at the higher end of the spectral leading edge 712 of emission spectrum 710. Although emission spectra 702, 710 are shown, the spectra can also be excitation spectra.

[0128] In one embodiment, more than three original images can be obtained. In one embodiment, each of the original images is used to analyze one and only one detection moiety. In one embodiment, one or more of the original images is used to analyze at least two of the detection moieties (i.e., there is overlap). In one embodiment, none of the original images are the same between the first detection moiety and the second detection moiety (i.e., all images are different). In one embodiment, at least one of the original images for the first detection moiety and at least one of the original images for the second detection moiety are the same image.

[0129] In one embodiment, an original image taken at the higher end of a spectral trailing edge can include the higher end of a spectral leading edge, and vice versa (i.e., an original image taken at the higher end of a spectral leading edge can include the higher end of a spectral trailing edge). In one embodiment, an 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., an original image taken at the higher end of a spectral trailing edge does not include the higher end of a spectral leading edge; or an original image at the higher end of a spectral leading edge does not include the higher end of a spectral trailing edge).

[0130] To identify the first detection moiety (as shown by emission spectrum 702) and the second detection moiety (as shown by emission spectrum 710), the raw images I3, I4, and I5 are analyzed and the relative contributions of the first detection moiety and the second detection moiety are determined. For example, the relative contributions can be determined by any appropriate mathematical, computational, or algebraic process or transformation, including but not limited to subtraction, differentiation, integration, etc., or combinations thereof. Then, based on the analysis of the raw images I3 and I4 (such as the relative contribution of the first detection moiety on the raw images I3 and I4), a final image of the first detection moiety is provided. Then, based on the analysis of the raw images I4 and I5 (such as the relative contribution of the second detection moiety on the raw images I4 and I5), a final image of the second detection moiety is provided.

[0131] Figure 9B Example first and second emission spectra similar to the first and second emission spectra of Figure 9A are shown, except that raw images I6, I7, and I8 have been obtained. Raw images I6 and I8 are taken at points where the emission intensity of the first detection moiety has the same or substantially the same value, and the emission intensity of the second detection moiety is different for each image. Raw image I7 is taken at a point such that at least three data points of the second detection moiety form a straight line.

[0132] Figure 9C Example first and second emission spectra similar to the first and second emission spectra of Figure 9A are shown, except that raw images I9 and I 10 are obtained. Raw image I9 is taken at a point where the higher end of the spectral trailing edge of the first detection moiety overlaps the lower end of the spectral leading edge of the second detection moiety. Raw image I 10 is taken at a point where the lower end of the spectral trailing edge of the first detection moiety overlaps the higher end of the spectral leading edge of the second detection moiety. As shown in Figure 9C , the trailing edge of the first detection moiety overlaps the leading edge of the second detection moiety such that raw image I9 includes the higher end of the spectral trailing edge of the first detection moiety and the lower end of the spectral leading edge of the second detection moiety, and raw image I 10 includes the lower end of the spectral trailing edge of the first detection moiety and the higher end of the spectral leading edge of the second detection moiety. In one embodiment, the leading edge of the first detection moiety overlaps the trailing edge of the second detection moiety.

[0133] Figure 9D Example first and second emission spectra similar to the first and second emission spectra of Figure 9C are shown, except that raw images I 11 -I 14 are obtained. Raw image I12 and I 13 The peak value of the spectrum can be used to determine the relative contribution of the detection moiety between the individual raw images, where the other raw image is at the spectral edge (leading or trailing edge) of the emission spectrum of the detection moiety for which the peak value of the emission was acquired.

[0134] In one embodiment, two or more raw images of the first emission spectrum are obtained, where at least one of the images is at the lower end of a spectral edge of the first emission spectrum and at least one of the images is at the upper end of the same spectral edge of the first emission spectrum. Two or more raw images of the second emission spectrum are obtained, where at least one of the images is at the lower end of a spectral edge of the second emission spectrum and at least one of the images is at the upper end of the same spectral edge of the second emission spectrum. A first final image of the first detection moiety (as depicted by the first emission spectrum) and a second final image of the second detection moiety (as depicted by the second emission spectrum) are provided, where the first final image and the second final image are based on the raw images from the first detection moiety and the second detection moiety. In one embodiment, at least one of the raw images of the first emission spectrum and the second emission spectrum is the same image. For example, the second image of the first emission spectrum (at the upper end of the spectral edge of the first emission spectrum) is the same image as the first image of the second emission spectrum (at the lower end of the spectral edge of the second emission spectrum).

[0135] Although two detection moieties are discussed, the process can be used for any number of detection moieties. In other words, two or more raw images of the nth emission / excitation spectrum are obtained, where at least one of the images is at the lower end of a spectral edge of the nth emission / excitation spectrum and at least one image is at the upper end of the spectral edge of the nth emission / excitation spectrum, and where n is greater than or equal to 1 (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). The process is then repeated for at least one or more emission / excitation spectra.

[0136] In one embodiment, to determine the signal contribution of each detector group with overlapping spectra, at least three data points for one detector group are obtained such that these three data points form a curve; and at least two data points for another detector group are obtained such that these two data points form a straight line. Which detector group requires data points to form a curve or a straight line is determined based on relative spectral edges. In other words, when using the same spectral edge (i.e., leading edge or trailing edge) of different emission spectra, an emission spectrum in which at least a portion of the same spectral edge falls below the emission spectrum of another emission spectrum requires only at least two data points. At least two data points (i.e., those data points forming a straight line) can be used to determine the contribution of a detector group, regardless of whether a curve provided by other detector groups exists or not; and additionally, at least three data points (i.e., those data points forming a curve) can be used to determine the contribution of other detector groups, regardless of whether a straight line provided by the initial detector group exists or not.

[0137] In one embodiment, to determine the signal contribution of each detection group with overlapping spectra, at least three data points for one detection group are acquired such that these three data points form a curve; and at least three data points for another detection group are acquired such that these three data points form a curve or a straight line. For example, refer back to the previous section. Figure 9D B 12 -B 14 The data points can be incorporated into the following formula:

[0138]

[0139] Among them, C B It is the curvature of emission B (e.g., the second derivative), S B12 Is it emission B in image I? 12 The signal strength at the wavelength S B13 Is it emission B in image I? 13 The signal strength at the wavelength, and S B14 Is it emission B in image I? 14 The signal strength at the wavelength. A 12 -A 14 The data points can be incorporated into the following formula:

[0140]

[0141] Among them, C A S is the curvature of emission A (e.g., the second derivative). A12 Is it emission A in image I? 12 The signal strength at the wavelength S A13 Is it emission A in image I? 13 The signal strength at the wavelength, and S A14Is it emission A in image I? 14 The signal strength at the wavelengths. Because of the stronger curvature (i.e., more positive (e.g., +5 stronger than +2; as another example, +4 stronger than -1) or more negative (e.g., -6 stronger than -1; as another example, -5 stronger than +2) at these emission wavelengths corresponding to each detection group, the detection groups are distinguishable from each other. In other words, the detection groups have stronger curvature at different emission wavelengths. For example, at the same emission wavelength, Alexa 647 has a stronger curvature at 660nm, 670nm, and 680nm than Alexa 594. At the same emission wavelength, Alexa 594 has a stronger curvature at 609nm, 619nm, and 632nm than Alexa 647. Therefore, Alexa 647 can be distinguished from Alexa 594 based on the curvature at 660nm, 670nm, and 680nm; and Alexa 594 can be distinguished from Alexa 647 based on the curvature at 609nm, 619nm, and 632nm.

[0142] Figure 9E Four images I were depicted 15 -I 18 The three emission spectra within are 702, 710, and 720 (A, B, C). The leading edge of emission B is located below the leading edge of emission A. Therefore, at least three data points of emission A were obtained (e.g., A). 15 -A 17 ; or A 15 A 16 A 18 ), and obtained at least two data points of transmission B (e.g., B). 15 and B 16 ; or B 15 and B 17 ; or B 16 and B 17 Additionally, the leading edge of emitter C is located below the leading edge of emitter B. Therefore, at least three data points of emitter B were obtained (e.g., B...). 15 B 17 B 18 ; or B 16 -B 18 ), and obtained at least two data points of emission C (e.g., C 16 and C 17 ; or C 17 and C 18 ; or C 16 and C 18 The corresponding data points from the emission AC can be used to determine the corresponding contribution of the detection group.

[0143] In one embodiment, two or more raw images are obtained at two different emission wavelengths of a first emission spectrum, wherein at least one of the images is located at the spectral leading edge or spectral trailing edge of the first emission spectrum, and at least one of the images is located at the spectral trailing edge or spectral leading edge of the first emission spectrum. In other words, two or more raw images are located at different spectral edges of the same emission spectrum (i.e., at least one raw image at the spectral leading edge and at least one raw image at the spectral trailing edge, wherein the spectral leading edge is located in the emission spectrum of a detection group).

[0144] like Figure 9E As shown, the signals of emission spectra A and B are obtained from the original images at different spectral edges of their emission spectra (A of emission spectrum A). 15 and A 17 ; and the emission spectrum B of B 15 / B 16 / B 17 and B 18 The intensity of signals at different spectral edges can be equal or unequal (e.g., the intensity can be larger at one spectral edge and smaller at another).

[0145] In one embodiment, two or more raw images of a first emission spectrum are obtained, wherein at least one of the raw images is located at the spectral leading edge or trailing edge of the first emission spectrum, at least one of the raw images is located at the spectral trailing edge or leading edge of the first emission spectrum, and at least one of the raw images is located at the peak intensity wavelength. In other words, two or more raw images are located at different spectral edges of the same emission spectrum, and one raw image is located at the peak intensity wavelength. Figure 9E As shown, emission spectrum A provides signals at different spectral edges of its emission spectrum (Ae of emission spectrum A). 15 and A 17 ) and the signal at the peak intensity wavelength (A of the emission spectrum A) 16 ).

[0146] although Figure 9E The original images obtained from emission spectrum A at the spectral leading edge, peak emission, and spectral trailing edge are shown, but this is not intended to be limited to a single emission spectrum. The same type of original images can be obtained for as many emission spectra as desired.

[0147] In one embodiment, changes in signal strength (i.e., pixel level) can be used to identify detection groups.

[0148] In one embodiment, such as when a representative point of the emission spectrum is obtained, the rate of change or change in signal intensity can be determined based on the trailing edge of the spectrum. In one embodiment, such as when a representative point of the emission spectrum is obtained, the rate of change or change in signal intensity can be determined based on the leading edge of the spectrum.

[0149] In one embodiment, such as when a representative point of the excitation spectrum is obtained, the rate of change or change in intensity can be determined based on the trailing edge of the spectrum. In one embodiment, such as when a representative point of the excitation spectrum is obtained, the rate of change or change in intensity can be determined based on the leading edge of the spectrum.

[0150] In one embodiment, the change in signal intensity can be compared to an expected value. For example, the change in intensity can be up to + / - 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, the change in signal intensity can be compared to a threshold value. In one embodiment, the change in signal intensity can be positive or negative, such that a positive or negative change identifies a desired detection moiety.

[0151] In one embodiment, a threshold value can be applied, such as during image processing and analysis, to determine whether a signal is caused by a desired detection moiety, an undesired detection moiety, noise, or background.

[0152] In one embodiment, when the change in signal intensity between a first image and a second image, such as at a desired or predetermined wavelength, is equal to or greater than a first threshold value, the pixel or signal is "kept on" for analysis for the resulting image; whereas when the change in signal between the first image and the second image is less than the first threshold value, the pixel or signal is "turned off" for analysis for the resulting image.

[0153] In one embodiment, a first emission derivative of the emission spectrum 702 of a first detection moiety can be obtained; and, a second emission derivative of the emission spectrum 710 of a second detection moiety can be obtained. Although a first derivative is discussed, any higher order derivative can be calculated when desired.

[0154] In one embodiment, such as when 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, such as when a representative point of the emission spectrum is obtained, the intensity change may be positive, positive at least a threshold amount, and / or a positive multiple of the first emission. In one embodiment, such as when 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 less than -3). In one embodiment, such as when a representative point of the excitation spectrum is obtained, the intensity change may be negative, negative at least a threshold amount, and / or negative a multiple of the first excitation.

[0155] As an example, Δx (change in emission wavelength) is 10 nm and Δy (change in emission intensity) is 50%, with a slope of 50% / 10 nm or 5% / nm. When comparing the first and second images, an increase in intensity of at least 5 times between corresponding pixels can be attributed to the first detection group, and the pixel is "held"; while an increase in intensity of less than 5 times between corresponding pixels can be attributed to factors other than the first detection group (e.g., background), and the pixel is "turned off". This example is not intended to be limited to values ​​and / or percentages. The first threshold may include a range based on expected or anticipated changes in emission intensity. For example, the first threshold can allow the slope to be + / - (added or subtracted) up to 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%.

[0156] The slope satisfies the condition given by the following formula:

[0157]

[0158] In one embodiment, when the first detection group is excited (and thus the first image is obtained), the wavelength of the first excitation light can be selected so as not to excite the second detection group. Then, the wavelength of the second excitation light can also be selected to excite the first detection group (thereby providing the second image) and not to excite the second detection group. The first and second images can be processed and compared to obtain the change in emission intensity (in other words, the slope or y / x) based on the emission of the first detection group due to the change in excitation wavelength.

[0159] In one embodiment, at least one of the excitation lights can stimulate one or more detection groups. However, as discussed below, the resulting slope can be used to remove the signal of one or more undesired detection groups.

[0160] In one embodiment, two or more images produced by two or more excitation wavelengths can be compared and processed to calculate a desired slope. The resulting slope can be used to keep the signal or turn off the signal in a final image. In one embodiment, two or more signals produced by two or more excitation wavelengths can be compared and processed to calculate a desired slope. The resulting slope can be used to keep the signal or turn off the signal in a final image.

[0161] In one embodiment, a first or higher order derivative can be calculated for each detection group spectral edge. In one embodiment, the spectral edge of the respective detection group can be used to distinguish the emission of different detection groups.

[0162] In one embodiment, the minimum number of raw images is n, where n is the number of detection groups. For example, a first raw image can be obtained at a higher end of a trailing edge of a first emission spectrum and a lower end of a leading edge of a second emission spectrum. A second raw image can be obtained at a lower end of the trailing edge of the first emission spectrum and a higher end of the leading edge of the second emission spectrum. The first and second raw images can be processed and / or analyzed to provide a first final image of a first detection group (as depicted by the first emission spectrum) and a second final image of a second detection group (as depicted by the second emission spectrum). Although emission spectra are discussed, this embodiment can be implemented on excitation spectra.

[0163] In one embodiment, the minimum number of raw images is n+1, where n is the number of detection groups.

[0164] In one embodiment, all raw images and final images of the first and second detection groups are displayed to an end user or operator such as on a screen (e.g., a screen of at least one of a phone, a tablet, a computer, a television, a PDA, a hand-held device, etc.). In one embodiment, at least one of the raw images of the first and / or second detection groups is displayed. In one embodiment, at least one of the final images of the first and / or second detection groups is displayed. In one embodiment, no raw images are displayed, but at least one of the final images is displayed. In one embodiment, no raw images are displayed, but all of the final images are displayed.

[0165] Embodiments for acquiring signals (e.g., same spectral edges, different spectral edges, peak and one spectral edge, peak and two spectral edges, etc.) are not intended to be limited to the emission spectra specifically discussed for the examples. Rather, signal acquisition can be applied to one or more emission spectra, where all emission spectra have the same acquisition (e.g., same spectral edges, different spectral edges, peak and one spectral edge, peak and two spectral edges, etc.), at least two emission spectra have the same acquisition, or no emission spectra have the same acquisition.

[0166] To obtain the raw images, imaging can be performed with a flow cytometer or a microscope, such as a fluorescence microscope, a scanner, etc. Imaging can be performed in conventional epi-fluorescence, light sheet microscopy, super-resolution microscopy, and confocal microscopy.

[0167] Any image or file (whether raw or processed) can be stored in any appropriate storage medium at any point during execution of any embodiment of the present application. Storage media include, but are not limited to, one or more of a hard disk, a random access memory (RAM), a read-only memory (ROM), a storage device of a distributed computing system, an optical disk (such as a compact disc, a digital versatile disc, or a Blu-ray disc), a flash memory device, a memory card, etc.

[0168] Embodiments of the present application include a non-transitory computer readable medium that can store instructions for performing the above-described methods and any steps thereof, including any combination thereof. For example, the non-transitory computer readable medium can store instructions for execution by one or more processors or similar devices.

[0169] Embodiments of the present application include two or more non-transitory computer readable media that can store instructions for performing the above-described methods and any steps thereof, including any combination thereof. For example, the instructions for execution can be split among two or more processors or similar devices.

[0170] Other embodiments of the present application can also include a computer or device (e.g., a phone, a tablet, a PDA, etc.) such as a non-transitory computer readable medium that reads and executes computer executable instructions recorded on the storage medium (which can be the same or different storage medium as the storage medium used to store the images or files as discussed above) to perform the functions of any of the embodiments. The computer can include one or more central processing units (CPUs), microprocessing units (MPUs) or other circuits that can include a standalone computer or networked computers. The computer executable instructions can be provided from a network or the storage medium to the computer, for example.

[0171] The computer or device can also be configured to display any of the images or files, whether original or processed, such as on a display or screen.

[0172] When a feature or element is referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and / or elements can also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected," "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element, or intervening features or elements can be present. In contrast, when a feature or element is referred to as being "directly connected," "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or illustrated with respect to one embodiment, the features and elements so described or illustrated can be applied to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature can have portions that overlap or underlie the adjacent feature.

[0173] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of 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. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and can be abbreviated as " / ".

[0174] For convenience in description, spatially relative terms, such as "beneath", "below", "lower", "above", "upper", and the like, can be used herein for the purpose of illustrating one element or feature's relationship to another element(s) or feature(s) as depicted in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device, system, or method depicted in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "below" can encompass both an orientation of above and below. The device can be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal", and the like are used herein for the purpose of explanation only unless specifically indicated otherwise. Additionally, "lower", "higher", and the like are used to depict such elements, features, information, etc. that are further down or further up on a graph, chart, or curve relative to one another or at least other elements, features, information, etc.

[0175] Although the terms "first" and "second" can be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms. These terms can be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element. Additionally, while the terms "first" and "second" can be used herein to describe various features / elements, these features / elements should not be limited by these terms. These terms can be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element. Additionally, although the terms "first" and "second" are used herein, these terms are not intended to limit various features / elements to only one or two. Rather, three (i.e., a third), four (i.e., a fourth), or more can be included or used, where appropriate or desired.

[0176] In this specification and the following claims, the word "comprise" and variations such as "comprising" and "comprises" means that various components can be employed collectively in a method and an article (e.g., compositions and devices including devices and methods). For example, the term "comprise" will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0177] As used herein in the specification and claims, including in the examples, and unless explicitly stated otherwise, all figures may be read as if they begin with the words “about” or “approximately”, even if the term is not explicitly stated. When describing magnitude and / or location, the phrases “about” or “approximately” may be used to indicate that the described value and / or location is within a reasonably expected range of value and / or location. For example, a numerical value may be + / - 0.1% of the value (or range of values), + / - 1% of the value (or range of values), + / - 2% of the value (or range of values), + / - 5% of the value (or range of values), + / - 10% of the value (or range of values), etc. Unless the context otherwise indicates, any numerical value given herein should also be understood to include approximately or approximately that value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range stated herein is intended to include all subranges contained therein. It should also be understood that, as properly understood by one of those skilled in the art, when a value is disclosed, the terms "less than or equal to" that value, "greater than or equal to" that value, and possible ranges between values ​​are also disclosed. For example, if the value "X" is disclosed, then "less than or equal to X" and "greater than or equal to X" (e.g., where X is a numerical value) are also disclosed. It should also be understood that throughout the application, data is provided in various different formats, and this data represents a range of any combination of endpoints and start points, as well as data points. For example, if specific data point "10" and specific data point "15" are disclosed, it should be understood that values ​​greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15, as well as values ​​between 10 and 15, are considered disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0178] While various exemplary embodiments have been described above, any of a variety of variations may be made to the various embodiments without departing from the scope of the invention as described in the claims. For example, in alternative embodiments, the order in which the various method steps described are performed may often be changed, while in other alternative embodiments, one or more method steps may be skipped together. In some embodiments, optional features of various device and system embodiments may be included, while in other embodiments they may not be included. Therefore, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

[0179] The examples and illustrations included herein are shown by way of illustration and not limitation of specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived therefrom, allowing for structural and logical substitutions and changes without departing from the scope of this disclosure. For convenience only, the term "inventory" may be used herein, individually or collectively, to refer to these embodiments of the inventive subject matter, without intending to voluntarily limit the scope of this application to any single invention or inventive concept in the event that more than one invention or inventive concept is disclosed. Therefore, while specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may replace the specific embodiments shown. This disclosure is intended to cover any and all modifications or variations of the various embodiments. After reading the above specification, combinations of the above embodiments and other embodiments not specifically described herein will be clear to those skilled in the art.

[0180] For purposes of explanation, specific nomenclature has been used in the foregoing description to provide a thorough understanding of this disclosure. However, it will be clear to those skilled in the art that these specific details are not necessary for practicing the systems and methods described herein. For purposes of illustration and description, the foregoing description of specific embodiments has been presented by way of example. These are not intended to be exclusive or to limit the disclosure to the precise forms described. Many modifications and variations are possible in light of the foregoing teachings. The embodiments are shown and described in order to best explain the principles and practical application of this disclosure, thereby enabling others skilled in the art to best utilize this disclosure and various embodiments with various modifications suitable for the particular intended use. The scope of this disclosure is intended to be defined by the following claims and their equivalents.

Claims

1. An imaging system comprising: at least one tiltable filter assembly comprising an optical filter configured to be disposed in an optical path of the imaging system; a dynamic correction optic configured to be disposed in the optical path of the imaging system; and a fixed correction optic configured to be disposed in the optical path of the imaging system, wherein the optical filter and the dynamic correction optic are configured to tilt in a substantially parallel X-axis, and the fixed correction optic is tilted in a substantially perpendicular Y-axis; and wherein, for a selected first angle of incidence of the optical filter, the dynamic correction optic is configured to tilt to a second angle of incidence, and wherein an angle of incidence of the fixed correction optic is configured to produce astigmatism to mitigate astigmatism caused by a combination of the optical filter at the selected first angle of incidence and the dynamic correction optic at the second angle of incidence.

2. The imaging system of claim 1, further comprising a telecentric tube lens configured to be disposed in the optical path of the imaging system. Any residual astigmatism caused by a combination of the fixed correction optic, the dynamic correction optic, and the optical filter is approximately zero (0).

3. The imaging system of claim 1, wherein, The dynamic correction optic is configured to stabilize lateral image shift caused by tilting of the optical filter.

4. The imaging system of claim 1, wherein, A sum of lateral image shift caused by the dynamic correction optic and lateral image shift caused by tilting of the optical filter is substantially constant.

5. The imaging system of claim 4, wherein, For a selected first angle of incidence of the optical filter, the dynamic correction optic is configured to tilt to a second angle of incidence, and wherein a sum of lateral image shift caused by the dynamic correction optic and lateral image shift caused by the optical filter is substantially constant.

6. The imaging system of claim 4, wherein, The telecentric tube lens is located at a position in the optical path of the imaging system where the telecentric tube lens is telecentric in both image and object space.

7. The imaging system of claim 2, wherein, 8. The imaging system of claim 1, further comprising a filter translator configured to hold the at least one tiltable filter assembly. The filter translator is a filter wheel, and wherein the at least one tiltable filter assembly is configured to tilt the optical filter to an angle of incidence selected from a range of 0° to 89.9°.

9. The imaging system of claim 8, wherein, The low incidence filter is configured to have an angle of incidence of excitation light selected from a range of 10.0° to 30.0°.

10. The imaging system of 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 reject filter, and a multi-pass filter, wherein, The imaging system is a fluorescence microscope imaging system.

11. The imaging system of claim 1, wherein, The optical filter is an interference filter.

12. The imaging system of claim 1, wherein, 13. A method of mitigating astigmatism in an imaging system, the method comprising: disposing an optical filter in an optical path of the imaging system at a first angle of incidence; disposing a dynamic correction optic in the optical path of the imaging system at a second angle of incidence; disposing a fixed correction optic in the optical path of the imaging system at a third angle of incidence, ​ wherein the optical filter and the dynamic correction optic are configured to tilt in a substantially parallel X-axis, and the fixed correction optic is tilted in a substantially perpendicular Y-axis; and wherein an angle of incidence of the fixed correction optic is configured to produce astigmatism to mitigate astigmatism caused by a combination of the optical filter at the first angle of incidence and the dynamic correction optic at the second angle of incidence.

Images