Patent Description:
Samples often include materials of interest that are to be imaged for analysis. These materials of interest may include a plurality of biomarkers and/or components for which it may be desirous to detect and image. Current filters and imaging apparatuses may only permit for a limited number of labels to be used at any one given time. As a result, practitioners, researchers, and those working with suspensions continue to seek systems and methods to more efficiently and accurately image samples.

<CIT> discloses methods comprising staining with nano-crystalline luminescent semiconductor material called quantum dots.

<CIT> discloses methods of determining the concentration of fluorescent pigments in a sample.

Further aspects and preferred embodiments of the invention are defined in the dependent claims. Any aspects, embodiments and examples of the present disclosure which do not fall under the scope of the appended claims are provided for illustrative purposes.

This disclosure is directed to a system and method for obtaining spectral data of individual detection moieties from a plurality of detection moieties, such as those detection moieties having overlapping spectra, based on emission and/or excitation wavelengths of the respective detection moieties used. This disclosure is further directed to a system and method for recording fluorescent images while distinguishing between multiple separate fluorescent targets.

In the following description, the term "raw image" is used to describe an image (whether or not visually displayed to an operator or end user) including data or at least one signal, having been captured by a sensor or detector, which has not been processed.

In the following description, the term "final image" is used to describe an image (whether or not visually displayed to an operator or end user) including data or at least one signal which has been processed. "Final image" can also be used to describe an image (whether or not visually displayed to an operator or end user) which is an output image resulting from the comparison and/or analysis of two or more raw or other final images.

In the following descriptions, the term "light" is used to describe various uses and aspects of multiplexing and imaging. 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 and infrared portions of the electromagnetic spectrum.

In the following descriptions, the term "sample" is used to describe a biological fluid, a biological semi-solid, a biological solid (which may remain solid, such as tissue, or may be liquefied in any appropriate manner), a suspension, a portion of the suspension, a component of the suspension, or the like.

In the following descriptions, the terms "target analyte" or "target material" are used to describe a biological material of interest.

In the following descriptions, the term "non-target analyte" is used to describe a biological material which is not a target analyte.

In the following descriptions, the term "biomarker" is used to describe a substance that is present on or within the target analyte or target material (i.e. intracellular or extracellular the target analyte; internalized, such as through phagocytosis, within the target analyte; or the like). Biomarkers include, but are not limited to, peptides, proteins, subunits, domains, motifs, epitopes, isoforms, DNA, RNA, or the like. The biomarker may be a target molecule for drug delivery.

In the following descriptions, the term "affinity molecule" is used to describe any molecule that is capable of binding to or interacting with another molecule. The interaction or binding can be covalent or non-covalent. The affinity molecule includes, but is not limited to, an antibody, a hapten, a protein, an aptamer, an oligonucleotide, a polynucleotide, or any appropriate molecule for interacting with or binding to another molecule (e.g., a biomarker; a molecule of a binding pair or a complementary molecule, including, without limitation, biotin or an avidin; or, the like).

In the following descriptions, the term "detection moiety" is used to describe a compound or substance which provides a signal for detection, thereby indicating the presence of another compound or substance, an analyte, or the like within a sample or specimen. The detection moiety 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, non-protein organic molecules, a nanoparticle (e.g., nanodiamond), or the like.

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

The chromogenic dye, which can be used with various enzyme labels (e.g. horseradish peroxidase and alkaline phosphate), includes, but is not limited to, <NUM>,<NUM>'-Diaminobenzidine (DAB), <NUM>-Amino-<NUM>-Ethylcarbazole (AEC), <NUM>-Chloro-<NUM>-Naphtol (CN), P-Phenylenediamine Dihydrochloride/pyrocatechol (Hanker-Yates reagent), Fast Red TR, New Fuchsin, Fast Blue BB, or the like. Fluorescent probes include, but are not limited to <NUM>,<NUM> IAEDANS; <NUM>,<NUM>-ANS; <NUM>-Methylumbelliferone; <NUM>-carboxy-<NUM>,<NUM>-dichlorofluorescein; <NUM>-Carboxyfluorescein (<NUM>-FAM); <NUM>-Carboxynapthofluorescein; <NUM>-Carboxytetramethylrhodamine (<NUM>-TAMRA); <NUM>-FAM (<NUM>-Carboxyfluorescein); <NUM>-HAT (Hydroxy Tryptamine); <NUM>-Hydroxy Tryptamine (HAT); <NUM>-ROX (carboxy-X-rhodamine); <NUM>-TAMRA (<NUM>-Carboxytetramethylrhodamine); <NUM>-Carboxyrhodamine <NUM>; <NUM>-CR <NUM>; <NUM>-JOE; <NUM>-Amino-<NUM>-methylcoumarin; <NUM>-Aminoactinomycin D (<NUM>-AAD); <NUM>-Hydroxy-<NUM>-methylcoumarin; <NUM>-Amino-<NUM>-chloro-<NUM>-methoxyacridine; ABQ; Acid Fuchsin; ACMA (<NUM>-Amino-<NUM>-chloro-<NUM>-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AutoFluorescent Protein; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™;.

Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alexa Fluor <NUM>™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red <NUM>; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow <NUM> GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO <NUM>(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H; Blue Fluorescent Protein); BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO™ -<NUM>; BOBO™ -<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>; Bodipy <NUM>/<NUM>-X; Bodipy <NUM>/<NUM>-X; Bodipy <NUM>/<NUM>; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -<NUM>; BO-PRO™ -<NUM>; Brilliant Sulphoflavin FF; Brilliant Violet <NUM>; Brilliant Violet <NUM>; Brilliant Violet <NUM>; Brilliant Violet <NUM>; Brilliant Violet <NUM>; Brilliant Violet <NUM>; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-<NUM>; Calcium Green-<NUM>; Calcium Green-5N; Calcium Green-C18; Calcium Orange; Calcofluor White; Carboxy-X-hodamine (<NUM>-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CF405S; CF488A; CF <NUM>; CF <NUM>; CF <NUM>; CF <NUM>; CF <NUM>; FP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3. <NUM><NUM>; Cy3. <NUM>™; Cy3™; Cy5. <NUM><NUM>; Cy5. <NUM>™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; DAPI; Dapoxyl; Dapoxyl <NUM>; Dapoxyl <NUM>; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine <NUM>); Di-<NUM>-ANEPPS; Di-<NUM>-ANEPPS; DiA (<NUM>-Di-<NUM>-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DiIC18(<NUM>)); DIDS; Dihydorhodamine <NUM> (DHR); DiI (DiIC18(<NUM>)); Dinitrophenol; DiO (DiOC18(<NUM>)); DiR; DiR (DiIC18(<NUM>)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-<NUM>-NHS; DY-<NUM>-NHS; EBFP (Enhanced Blue Fluorescent Protein); ECFP (Enhanced Cyan Fluorescent Protein); EGFP (Enhanced Green Fluorescent Protein); ELF <NUM>; Eosin; ER-Tracker™ Green; ER-Tracker™ Red; ER-Tracker™ Blue-White DPX; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-<NUM> (EthD-<NUM>); Euchrysin; EukoLight; Europium (III) chloride; EYFP (Enhanced Yellow Fluorescent Protein); Fast Blue; FDA; FIF (Formaldehyde Induced Fluorescence); FITC; FITC Antibody; Flazo Orange; Fluo-<NUM>; Fluo-<NUM>; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM <NUM>-<NUM>™; FM <NUM>-<NUM>; Fura Red™ (high pH); Fura Red™/Fluo-<NUM>; Fura-<NUM>, high calcium; Fura-<NUM>, low calcium; Fura-<NUM>/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink <NUM>; 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; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst <NUM>; Hoechst <NUM>; Hoechst <NUM>; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-<NUM>, high calcium; Indo-<NUM>, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf JC-<NUM>; JO-JO-<NUM>; JO-PRO-<NUM>; LaserPro; Laurodan; LDS <NUM>; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-<NUM>; LO-PRO-<NUM>; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-<NUM>; Mag-Fura-<NUM>; Mag-Indo-<NUM>; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin <NUM> GFF; Maxilon Brilliant Flavin <NUM> GFF; Merocyanin; Methoxycoumarin; Mitotracker Green; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); mStrawberry; NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green™; Oregon Green™ <NUM>; Oregon Green™ <NUM>; Oregon Green™ <NUM>; Pacific Blue; Pararosaniline (Feulgen); PBFI ; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5. <NUM>; PE-TexasRed (Red <NUM>); 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-<NUM>; POPO-<NUM>; PO-PRO-<NUM>; PO-PRO-<NUM>; Primuline; Procion Yellow; Propidium Iodid (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 <NUM>; Quinacrine Mustard; Red <NUM> (PE-TexasRed); Resorufin; RFP; RH <NUM>; Rhod-<NUM>; Rhodamine; Rhodamine <NUM>; Rhodamine <NUM>; Rhodamine <NUM> GLD; Rhodamine <NUM>; Rhodamine B; Rhodamine B <NUM>; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;.

Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin; rsGFP (red shifted GFP (S65T)); S65A ; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red <NUM>; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgGFP™ (super glow GFP; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-<NUM>; SNAFL-<NUM>; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (<NUM>-methoxy-N-(<NUM>-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTO <NUM>; SYTOX Blue; SYTOX Green; SYTOX Orange; SYTOX Red; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin <NUM>; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-<NUM>; TO-PRO-<NUM>; TO-PRO-<NUM>; TOTO-<NUM>; TOTO-<NUM>; TriColor (PE-Cy5); TetramethylRodamineIsoThioCyanate; True Blue; TruRed; Tubulin Tracker™ Green; Ultralite; Uranine B; Uvitex SFC; wt GFP (wild type GFP); WW <NUM>; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP (Yellow shifted); Green Fluorescent Protein; YFP (Yellow Fluorescent Protein); YO-PRO-<NUM>; YO-PRO-<NUM>; YOYO-<NUM>; YOYO-<NUM>; and, combinations and derivatives thereof. In one embodiment, the detection moiety, such as organic fluorophore, can have a molecule weight of at least <NUM> Daltons, including, without limitation, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, at least <NUM> kD, and at least <NUM> kD.

In the following descriptions, the terms "stain" or "label," which are used interchangeably, are used to describe an affinity molecule bound to or interacted with a detection moiety. The binding or interaction can be direct or indirect. Direct binding or interaction includes covalent or non-covalent interactions between the biomarker and the detection moiety. Indirect binding or interaction includes the use of at least first and second complementary molecules which form binding pairs. The first and second complementary molecules are, in combination, binding pairs which can bind or interact in at least one of the following manners: hydrophobic interactions, ionic interactions, hydrogen bonding interactions, non-covalent interactions, covalent interactions, affinity interactions, or the like. The binding pairs include, but are not limited to, immune-type binding-pairs, such as, antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or primary antibody-secondary antibody; nonimmune-type binding-pairs, such as biotin-avidin, biotin-streptavidin, folic acid-folate binding protein, hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme-substrate analog, enzyme-pseudo-substrate (substrate analogs that cannot be catalyzed by the enzymatic activity), enzyme-cofactor, enzyme-modulator, enzyme-inhibitor, or vitamin B <NUM>-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 linkers (e.g., polyethylene glycol); or polynucleotide-polynucleotide binding protein. The binding pairs can be included within or used as amplification techniques. Amplification techniques are also implemented to increase the number of detection moieties bound to or interacted with the biomarker to increase a signal. In one embodiment, when binding pairs are used, the stain can be pre-conjugated, such that, during a labeling, staining, or adding step, the affinity molecule is already bound to or interacted with a detection moiety when added to the sample. In one embodiment, when binding pairs are used, the stain can be conjugated in the sample, such that the labeling, staining, or adding step includes at least two sub-steps including introducing (in any desired or appropriate order) an affinity molecule-first binding molecule conjugate and a second binding pair molecule-detection moiety conjugate, wherein the first and second binding pair molecules are complementary and bind to or interact with each other.

Furthermore, "a plurality of stains" can be used to describe two or more stains in which the affinity molecules and/or the detection moieties are different. For example, anti-CK-Alexa <NUM> is different than anti-EpCAM-Alexa <NUM>. As another example, anti-CK-Alexa <NUM> is different than anti-CK-Alexa <NUM>.

In the following descriptions, the term "conjugate" is used to describe a first chemical, molecule, moiety, or the like bound to or interacted with a second chemical, molecule, moiety, or the like. The binding or interaction is direct or indirect. Direct binding or interaction includes covalent or non-covalent interactions between the biomarker and the detection moiety. Indirect binding or interaction includes the use of at least first and second complementary molecules which form binding pairs. The first and second complementary molecules are, in combination, binding pairs which binds or interacts in at least one of the following manners: hydrophobic interactions, ionic interactions, hydrogen bonding interactions, non-covalent interactions, covalent interactions, affinity interactions, or the like. The binding pairs include, but are not limited to, immune-type binding-pairs, such as, antigen-antibody, antigen-antibody fragment, hapten-anti-hapten, or primary antibody-secondary antibody; nonimmune-type binding-pairs, such as biotin-avidin, biotin-streptavidin, folic acid-folate binding protein, hormone-hormone receptor, lectin-specific carbohydrate, enzyme-enzyme, enzyme-substrate, enzyme-substrate analog, enzyme-pseudo-substrate (substrate analogs that cannot be catalyzed by the enzymatic activity), enzyme-cofactor, enzyme-modulator, 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 linkers (e.g., polyethylene glycol); or polynucleotide-polynucleotide binding protein.

In the following description, the term "signal" is used to describe an electric current or electromagnetic field which conveys 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 the detection moiety on or within a target analyte, such as a cell.

In the following description, the term "multiplex" is used to describe process or kit by which a sample is labeled with a plurality of stains. Each of the detection moieties emit different wavelengths. For example, at least two stains can be used to label the sample. Multiplexing can include up to <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more stains.

An example method for labeling a biomarker on a target analyte is discussed. In one embodiment, a sample, suspected of including at least one target analyte is obtained. Suitable devices, systems, and/or methods of sample collection and/or processing may include those described in one or more of the following <CIT>; <CIT>; <CIT>;<CIT>; <CIT>;<CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>;<CIT>; <CIT>. Suitable devices, systems, and/or methods for target analyte retrieval, isolation, or picking may include those described in one or more of the following <CIT>; <CIT>; <CIT>;<CIT>; <CIT>; <CIT>.

In one embodiment, the sample can undergo staining after collection of the sample. In one embodiment, the sample can undergo staining after processing the sample. In one embodiment, the sample can be multiplexed. At least one stain is added to the sample for labeling, such as by an autostainer or manually by an operator. In one embodiment, the 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, the at least one target analyte and the at least one non-target analyte or materials are stained.

After staining, the sample can be imaged, whereby the stained sample is illuminated with one or more wavelengths of excitation light, such as infrared, red, blue, green, and/or ultraviolet, from a light source, such as a laser or a light-emitting diode. The imaging can be done with a flow cytometer or a microscope, such as a fluorescent microscope, a scanner, or any other appropriate imaging system or modality. In one embodiment, imaging can be performed in a system in which a detection moiety, when imaged, can provide a signal across a spectrum, including, without limitation, brightfield and/or darkfield illumination, fluorescence, and the like. The images formed can be overlaid when a plurality of detection moieties is used. Emission, reflection, diffraction, scatter, and combinations thereof are used in for detection/imaging. The images can be analyzed to detect, enumerate, and/or locate the target analyte, such as when it is desirous to retrieve or pick the target analyte. Imaging is performed in a tube, on a microscope slide, or in any appropriate vessel or substrate for imaging.

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 method can be performed by any combination of the above. The methods described can be used in a system in which a detection moiety, when imaged, can provide a signal across a spectrum, including, without limitation, brightfield and/or darkfield illumination, fluorescence, and the like.

Spectral edge detection is process by which components of a sample are distinguished from other components of the sample by determining the contribution of the component of the sample within one or more signals. For example, the sample component can be a detection moiety, a chemical, a biomolecule, a structure, a compound, a substance, a deposit, the like, or combinations thereof. In other words, spectral edge detection is process by components having an emission spectrum can be distinguished from other components.

In one aspect, spectral edge detection is a process by which individual detection moieties can be distinguished from a plurality of detection moieties (i.e., during multiplexing), such as by distinguishing the detection moieties orthogonally-in that there is no ambiguity as to which detection moiety is being detected and/or imaged. A feature of a signal of interest, such as from a detection moiety of interest, can be distinguished in the presence of a featureless signal (i.e., the signal has an unknown value and/or structure), such as from background, autofluorescence, or a non-desired detection moiety. In other words, spectral edge detection determines the contribution of a detection moiety of interest across a plurality of signals (or images) composed of contributions from a plurality of detection moieties having at least partially overlapping spectra by eliminating the contributions from the non-desired detection moieties across the plurality of the signals (or images) when the intensities (and therefore the respective contributions) of the detection moieties are unknown.

Raw images are acquired at a given wavelength, for example, at a given emission wavelength, of the emission spectra. Each raw image includes a total signal. Each total signal comprises one or more signals from one or more detection moieties. Each total signal can further comprise a signal due to background or autoflurescence.

Spectral edge detection can also account for minor changes in signals, for example, when detecting a detection moiety against a reference detection moiety or when incorporating detection moieties which have emission shifts based on one or more factors, whether intentional (e.g., detection moieties which detect sample variables, including, for example, oxygen concentration, metal ion concentation, environmental changes, being endocytossed, eing exocytosed, or the like) or whether unintentional (e.g., variable pH of the sample or reagents causes detection moieties to have an emission shift).

Spectral edge detection uses an edge (e.g., a trailing edge or a leading edge) of an emission or excitation spectrum, such as for a detection moiety, to identify a single detection moiety within a plurality of spectrally-overlapping detection moieties. For example, two raw images can be obtained along the same leading or trailing spectral edge of the detection moiety; two raw images can be obtained along different leading and trailing spectral edges of the detection moiety; or, one raw image can be obtained along the leading or trailing spectral edge of the detection moiety and one raw image can be obtained at the peak emission of the detection moiety. Spectral edge detection can also utilize a combination of the implementations discussed above for a single detection moiety or for a plurality of detection moieties. For example, when using a plurality of detection moieties, a first detection moiety can be detected with signals from a peak and a leading spectral edge; a second detection can be detected with signals from a leading spectral edge; a third detection can be detected with signals from a leading spectral edge and a trailing spectra edge. Furthermore, spectral edge detection can form a curve for a first detection moiety and a line for a second detection moiety, such that at least a portion of the line falls under the curve, with data points of each emission spectrum (for example, signals at given emission/excitation wavelengths).

<FIG> shows an emission spectrum <NUM> of a first detection moiety. The emission spectrum <NUM> includes a leading spectral edge <NUM> and a trailing spectral edge <NUM>. In other words, the leading spectral edge <NUM> is a portion of the emission spectrum <NUM> to the left of a peak emission <NUM>; the trailing spectral edge <NUM> is a portion of the emission spectrum <NUM> to the right of the peak emission <NUM>. Though the emission spectrum <NUM> is shown, the spectrum can also be an excitation spectrum.

<FIG> shows an emission spectrum <NUM> of a second detection moiety. The emission spectrum <NUM> includes a leading spectral edge <NUM> and a trailing spectral edge <NUM>. In other words, the leading spectral edge <NUM> is a portion of the emission spectrum <NUM> to the left of a peak emission <NUM>; the trailing spectral edge <NUM> is a portion of the emission spectrum <NUM> to the right of the peak emission <NUM>. Though the emission spectrum <NUM> is shown, the spectrum can also be an excitation spectrum.

<FIG> shows an emission spectrum <NUM> of a third detection moiety. The emission spectrum <NUM> includes a leading spectral edge <NUM> and a trailing spectral edge <NUM>. In other words, the leading spectral edge <NUM> is a portion of the emission spectrum <NUM> to the left of a peak emission <NUM>; the trailing spectral edge <NUM> is a portion of the emission spectrum <NUM> to the right of the peak emission <NUM>. Though the emission spectrum <NUM> is shown, the spectrum can also be an excitation spectrum.

In one embodiment, any of the methods or systems can be used to detect a stain or detection while removing background or autofluorescence from an image or a 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 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 based on the raw images from the first detection moiety, and the first final image does not include the signal caused by the autofluorescence or background. This can be performed for any number of detection moieties to remove background or autofluorescence from any images.

In <FIG> and <FIG>, a fourth detection moiety (as depicted by emission spectrum <NUM>) is used as an example for the method by which an individual detetion moiety is distinguished over background or autofluorescnece. It should be noted, however, that the method discussed herein is not so limited and can also be implemented on the first, second, and/or third detection moieties (as depicted by emission spectra <NUM>, <NUM>, <NUM>) or any other appropriate detection moiety.

<FIG> shows the emission spectrum <NUM> and a background signal <NUM>. The background signal <NUM> is expected to be relatively unvarying with respect to the signal of interest and therefore depicted as a constant (i.e., a straight line) with a known value. Additionally, the relative intensities between the emission spectrum <NUM> and the background signal <NUM> are unknown.

For clarification purposes, <FIG> depict images Ii-Iis obtained from of a single wavelength. However, the images I<NUM>-I<NUM> can obtained across a given bandwidth (i.e., up to <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more nm), such that the images (as denoted by the dot-dot-dash lines) denote the average signal across the respective bandwidths. Additionally, though raw images are obtained, the signals (for example, the total signals comprising the individual signals) are from pixels in the raw images corresponding to an identical location of the sample between images. For example, Point A is a given location on or within the sample being imaged. A first pixel representing Point A in a first raw image comprises a first total signal. A second pixel representing Point A in a second raw image comprises a second total signal. Using one or more of the methods discussed herein, the first and second total signals of the first and second pixels of the first and second raw images, respectively, are assessed and/or compared to determine the contribution(s) of the individual detection moiet(ies).

<FIG> shows raw images I<NUM> and I<NUM> including first and second signals S<NUM> and S<NUM>, respectively, obtained during imaging. The signals S<NUM> and S<NUM> denote the total contributions of the fourth detection moiety and the background <NUM>. Raw image I<NUM> includes the first signal S<NUM> on a lower end of the leading spectral edge; and raw image I<NUM> is taken at a higher end of the leading spectral edge. To identify the fourth detection moiety (as shown by the emission spectrum <NUM>), the raw images I<NUM> and I<NUM>, are analyzed and the relative contribution of the fourth detection moiety between first and second signals S<NUM> and S<NUM> is determined by processing, comparing, and/or analyzing the change in signal with any appropriate mathematical, computational, or algebraic process or transformation, including, without limitation, subtraction, derivatives, or combinations thereof. A final image can then be provided depicting the fourth detection moiety based on the processing, comparing, and/or analyzing.

Spectral edge detection can be implemented for each detection moiety within the plurality of detection moieties, thereby allowing for multiplexing of a sample or fraction thereof 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 <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In one embodiment, any appropriate number of detection moieties can be used, including up to <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In one embodiment, any appropriate number of detection moieties can be used, including less than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

Spectral edge detection can be implemented for detetion moieties having spectral offsets, where the spectral offsets are spectral differences on the comparable spectral edge or at the spectral peaks. In one embodiment, this process can be implemented for detection moieties having differences in spectral offsets of less than or equal to <NUM>. In one embodiment, this process can be implemented for detection moieties having differences in spectral offsets of less than or equal to <NUM>. In one embodiment, this process can be implemented for detection moieties having differences in spectral offsets of <NUM>-<NUM>. In one embodiment, this process can be implemented for detection moieties having differences in spectra of <NUM>-<NUM>. In one embodiment, this process can be implemented for detection moieties having differences in spectral offsets of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In one embodiment, the difference between successive spectra (such as at the peak) can be the same (e.g., first and second detection moieties are separated by <NUM> and second and third detection moieties are separated by <NUM>). In one embodiment, the differences between successive spectra (such as at the peak) can be different (e.g., first and second detection moieties are separated by <NUM> and second and third detection moieties are separated by <NUM>).

In one embodiment, the signal contributions of each detection moiety (for example, by way of the contribution or subtraction coefficients) can be determined with at least two raw images, such as by cancelling out or nullifying the signal contributions provided by the non-interested detection moiety (i.e., a first detection moiety is the non-interested detection moiety and a second detection moiety is the detection moiety of interest; and/or, then the first detection moiety is the detection moiety of interest and the second detection moiety is the non-interested detection moiety) or background/autofluorescence. Any number of raw images equal to or greater than <NUM> can be obtained for spectral edge detection, including, without limitation, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more.

For clarity purposes regarding <FIG>, the emission spectrum <NUM> of the first detection moiety is also designated by "A"; the emission spectrum <NUM> of the second detection moiety is also designated by "B"; and, the emission spectrum <NUM> of the third detection moiety is also designated by "C. " The subscripts following A, B, or C in this description designate the image to which the detection moiety of interest is contributing intensity. For example, a data point A<NUM> denotes the contribution of A (or, the first detection moiety) within raw image I<NUM>, as shown by the data point A<NUM> on the emission spectrum <NUM>. So, A<NUM>-A<NUM> denote the contributions of A (or, the first detection moiety) in raw images I<NUM>-I<NUM>, respectively; B<NUM>-B<NUM> denote the contributions of B (or, the second detection moiety) in raw images I<NUM>-I<NUM>, respectively; and, C<NUM>-C<NUM> denote the contributions of C (or, the third detection moiety) in raw images I<NUM>-I<NUM>, respectively.

<FIG> shows the emission spectra <NUM>, <NUM> for the first and second detection moieties. Raw images I<NUM>, I<NUM>, and I<NUM> are obtained. Raw image I<NUM> is taken at a lower end of the leading spectral edge <NUM>; raw image I<NUM> is taken at a higher end of the leading spectral edge <NUM>, which also overlaps with a lower end of the leading spectral edge <NUM>; and, raw image I<NUM> is taken at a higher end of the leading spectral edge <NUM>. Though the emission spectra <NUM>, <NUM> are shown, the specta can also be excitation spectra.

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

In one embodiment, a raw image taken at the higher end of the trailing spectral edge can include the higher end of the leading spectral edge, and vice-versa (i.e., a raw image taken at the higher end of the leading spectral edge can include the higher end of the trailing spectral edge). In one embodiment, a raw image taken at the higher end of the particular spectral edge does not include the higher end of the opposing spectral edge (i.e., raw image at higher trailing spectral edge does not include higher leading spectral edge; raw image at higher leading spectral edge does not include higher trailing spectral edge).

To identify a first detection moiety (as shown by the emission spectrum <NUM>) and a second detection moiety (as shown by the emission spectrum <NUM>), the raw images I<NUM>, I<NUM>, and I<NUM> are analyzed and the relative contributions of the first and second detection moieties are determined. For example, the relative contributions can be determined by any appropriate mathematical, computational, or algebraic process or transformation, including, without limitation, subtraction, derivatives, or combinations thereof. A final image of the first detection moiety is then provided based on the analysis of raw images I<NUM>, I<NUM>, such as the relative contribution of the first detection moiety across the raw images I<NUM>, I<NUM>,. A final image of the second detection moiety is then provided based on the analysis of raw images I<NUM>, I<NUM>, such as the relative contribution of the second detection moiety across the raw images I<NUM>, I<NUM>.

<FIG> shows the example first and second emission spectra similar to that of <FIG>, except having obtained raw images I<NUM>, I<NUM>, and I<NUM>. Raw images I<NUM> and I<NUM> 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 between the images. Raw image I<NUM> is take at a point such that the at least three data points for the second detection moiety form a line.

<FIG> shows the example first and second emission spectra similar to that of <FIG>, except having obtained raw images I<NUM> and I<NUM>. Raw images I<NUM> and I<NUM> are taken at points where a higher end of a spectral edge of the first detection moiety overlaps with a lower end of a different spectral edge of the second detection moiety, and where a lower end of the spectral edge of the first detection moiety overlaps with a higher end of the different spectral edge of the second detection moiety. As shown in <FIG>, the trailing edge of the first detection moiety overlaps with the leading edge of the second detection moiety such that raw image I<NUM> includes the higher end of the trailing spectral edge of the first detection moiety and the lower end of the leading spectral edge of the second detection moiety, and raw image I<NUM> includes the lower end of the trailing spectral edge of the first detection moiety and the higher end of the leading spectral edge of the second detection moiety. In one embodiment, the leading edge of the first detection moiety overlaps with the trailing edge of the second detection moiety.

<FIG> shows the example first and second emission spectra similar to that of <FIG>, except having obtained raw images I<NUM>-I<NUM>. Raw images I<NUM> and I<NUM> are taken at the peaks of emission spectrum <NUM> and emission spectrum <NUM>, respectively. The peak emission of a spectrum can be used to determine the relative contribution of a detection moiety between raw images, wherein the other raw image is in a spectral edge (leading or trailing) of the emission spectrum of the detection moiety for which the peak emission is acquired.

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

Though two detection moieties are discussed, this process can be used for any number of detection moieties. In other words, two or more raw images of a nth emission/excitation spectrum are obtained, wherein at least one of the images is at a lower end of a spectral edge of the nth emission/excitation spectrum and at least one the images is at a higher end of the spectral edge of the nth emission/excitation spectrum, and wherein n is greater than or equal to <NUM> (i.e., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more). This process is then repeated for at least one more emission/excitation spectrum.

In one embodiment, to determine signal contribution by individual detection moieties having overlapping spectra, at least three data points of one detection moiety are to be obtained, such that the three data points form a curve; and, at least two data points of another detection moiety are to be obtained, such that the two data points form a line. The determination as to which detection moiety requires the data points to form the curve or the data points to form the line are based on the relative spectral edges. In other words, when using the same spectral edge (i.e., leading or trailing) of different emission spectra, the emission spectrum having at least a portion of the same spectral edge fall under the emission spectrum of the other emission spectrum only requires at least two data points. The at least two data points (i.e., those forming the line) can be used to determine the contribution of the detection moiety whether in the presence or absence of the curve provided by the other detection moiety; and, additionally, the at least three data points (i.e., those forming the curve—whether at least two are in the same spectral edge or whether one is at the peak intensity) can be used to determine the contribution of the other detection moiety whether in the presence or absence of the line provided by the initial detection moiety.

In one embodiment, to determine signal contribution by individual detection moieties having overlapping spectra, at least three data points of one detection moiety are acquired, such that the three data points form a curve; and, at least three data points of another detection moiety are acquired, such that the three data points form a curve or line. For example, in referring back to <FIG>, the data points of B<NUM>-B<NUM> can be incorporated into the following equation: <MAT> where CB is the curvature (e.g., second derivative) of emission B, SB12 is the signal intensity of emission B at the wavelength of image I<NUM>, SB13 is the signal intensity of emission B at the wavelength of image I<NUM>, and SB14 is the signal intensity of emission B at the wavelength of image I<NUM>. The data points of A<NUM>-A<NUM> can be incorporated into the following equation: <MAT> where CA is the curvature (e.g., second derivative) of emission A, SA12 is the signal intensity of emission A at the wavelength of image I<NUM>, SA13 is the signal intensity of emission A at the wavelength of image I<NUM>, and SA14 is the signal intensity of emission A at the wavelength of image I<NUM>. The detection moieties are distinguishable from each other because a stronger curvature (i.e., more positive (for example, +<NUM> is stronger than +<NUM>; as another example, +<NUM> is stronger than -<NUM>) or more negative (for example, -<NUM> is stronger than -<NUM>; as another example, -<NUM> is stronger than +<NUM>)) at those emission wavelengths corresponds to individual detection moieties. In other words, detection moieties have stronger curvatures across different emission wavelengths. For example, Alexa <NUM> has a stronger curvature across <NUM>, <NUM>, and <NUM> than Alexa <NUM> across the same emission wavelengths. Alexa <NUM> has a stronger curvature across <NUM>, <NUM>, and <NUM> than Alexa <NUM> across the same emission wavelengths. Therefore, based on the curvatures across <NUM>, <NUM>, and <NUM>, Alexa <NUM> can be distinguished from Alexa <NUM>; and, based on the curvatures across <NUM>, <NUM>, and <NUM>, Alexa <NUM> can be distinguished from Alexa <NUM>.

<FIG> depicts three emission spectra <NUM>, <NUM>, <NUM> (A, B, C) within four images I<NUM>-I<NUM>. A leading edge of emission B is underneath a leading edge of emission A. Therefore, at least three data points are obtained for emission A (e.g., A<NUM>-A<NUM>; or, A<NUM>, A<NUM>, A<NUM>), and at least two data points are obtained for emission B (e.g., B<NUM> and B<NUM>; or, B<NUM> and B<NUM>; or, B<NUM> and B<NUM>). Additionally, a leading edge of emission C is underneath a leading edge of emission B. Therefore, at least three data points are obtained for emission B (e.g., B<NUM>, B<NUM>, B<NUM>; or, B<NUM>-B<NUM>), and at least two data points are required for emission C (e.g., C<NUM> and C<NUM>; or, C<NUM> and C<NUM>; or, C<NUM> and C<NUM>). The respective data points for the emissions A-C can be used to determine the respective contributions of the detection moieties.

In one embodiment, two or more raw images at two distinct emission wavelengths of a first emission spectrum are obtained, wherein at least one of the images is in a leading spectral edge or a trailing spectral edge of the first emission spectrum and at least one of the images is in the trailing spectral edge or the leading spectral edge of the first emission spectrum. In other words, the two or more raw images are in different spectral edges of the same emission spectrum (i.e., at least one raw image in a leading spectral and at least one raw image in a trailing edge, wherein the leading spectral edges are in an emission spectrum of one detection moiety). As shown in <FIG>, signals of emission spectra A and B are acquired via raw images on different spectral edges of their emission spectra (A<NUM> and A<NUM> for emission spectrum A; and, B<NUM>/B<NUM>/B<NUM> and B<NUM> for emission spectrum B). The intensities of the signals on the different spectral edges can be equal or not equal (for example, the intensity can be greater on one spectral edge and less on the other spectral edge).

In one embodiment, two or more raw images of a first emission spectrum are obtained, wherein at least one of the raw images is in a leading spectral edge or a trailing spectral edge of the first emission spectrum, at least one of the raw images is in the trailing spectral edge or the leading spectral edge of the first emission spectrum, and at least one of the raw images is at the peak intensity wavelength. In other words, two or more raw images are in different spectral edges of the same emission spectrum and one raw image is at the peak intensity wavelength. As shown in <FIG>, emission spectrum A provides signals on different spectral edges of its emission spectra (A<NUM> and A<NUM> for emission spectrum A) and a signal at the peak intensity wavelength (A<NUM> for emission spectrum A).

Though <FIG> shows raw images acquired from emission spectrum A in the leading spectral edge, the peak emission, and the trailing spectral edge, this is not intended to be limited to only one emission spectrum. The same types of raw images can be acquired for as many emission spectra as desired.

In yet another aspect, chemicals, biomolecules, structures, compounds, substances, deposits, or the like can be distinguished, whether with or without being labeled or stained with a detection moiety. The non-detection moiety component, in a manner similar to the detection moieties discussed above, emits wavelengths when stimulated thereby forming an emission spectrum. The emission spectrum can be inherent to the non-detection moiety component, such that different non-detection moiety components have different emission spectra. The non-detection moiety components, however, comprise autofluorescent emission spectra. In other words, the non-detection moiety components need not be labeled or stained with detection moieties to provide a signal. The non-detection moiety components fluoresce due to natural properties inherent in their own composition, structure, or the like. The autofluorescent emission spectrum is used to distinguish the non-detection moiety component from detection moieties and other non-detection moiety components having overlapping emission spectra.

The same techniques, methods, and/or protocols discussed above and below can be applied to autofluorescent emission spectra, including those autofluorescent emission spectra provided by non-detection moiety components. Furthermore, as noted in <FIG>, the techniques, methods, and/or protocols discussed above and below can be applied to samples comprising detection moieties and non-detection moiety components.

Non-detection moiety components include, but are not limited to, nuclei, cytoplasm, cytosol, cell membrane, cell wall, fibers, cartilage, cells, interstitial fluid, proteins, glycoproteins, peptides, carbohydrates, nucleic acids, cytochromes, flavins, collagen, lipids, other biomolecules, intra-cellular and extra-cellular deposits, foreign materials, the like, and combinations thereof.

In one embodiment, change in signal intensity (i.e., pixel levels) can be used to identify a detection moiety.

In one embodiment, such as when representative points of the emission spectrum are obtained, the rate of change or the change in signal intensity can be determined based on the trailing edge of the spectrum. In one embodiment, such as when representative points of the emission spectrum are obtained, the rate of change or the change in signal intensity can be determined based on the leading edge of the spectrum.

In one embodiment, such as when representative points of the excitation spectrum are obtained, the rate of change or the change in intensity can be determined based on the trailing edge of the spectrum. In one embodiment, such as when representative points of the excitation spectrum are obtained, the rate of change or the change in intensity can be determined based on the leading edge of the spectrum.

In one embodiment, the change in signal intensity can be compared against an expected value. For example, the change in intensity can be the expected value +/- (plus or minus) up to <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%. In one embodiment, the change in signal intensity can be compared against a threshold. In one embodiment, the change in signal intensity can be positive or negative, such that the positive or negative change identifies the desired detection moiety.

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

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

In one embodiment, a first emission derivative of the emission spectrum <NUM> of the first detection moiety can be obtained; and, a second emission derivative of the emission spectrum <NUM> of the second detection moiety can be obtained. Though a first-order derivative is discussed, any higher-order derivative can be calculated when it is desirous to do so.

In one embodiment, such as when representative points of the emission spectrum are obtained, the rate of change can be greater than or equal to a threshold value. In one embodiment, such as when representative points of the emission spectrum are obtained, the change in intensity can be positive, positive by at least a threshold amount, and/or positive by a certain multiple of the first emission. In one embodiment, such as when representative points of the excitation spectrum are obtained, the rate of change can be less than or equal to a threshold value (i.e. more negative-for example -<NUM> is less than -<NUM>). In one embodiment, such as when representative points of the excitation spectrum are obtained, the change in intensity can be negative, negative by at least a threshold amount, and/or negative by a certain multiple of the first excitation.

As an example, Δx (the change in emission wavelength) is <NUM> and Δy (the change in emission intensity) is <NUM>%, the slope is <NUM>%/<NUM>, or <NUM>%/nm. When comparing the first and second images, an increase of intensity of at least <NUM> times between respective pixels can be attributed to the first detection moiety and the pixel is "kept on"; whereas an increase of intensity of less than <NUM> times between respective pixels can be attributed to something other than the first detection moiety (e.g., background) and the pixel is "turned off. " The example is not intended to be limited to values and/or percentages. The first threshold value can include a range based on the anticipated or expected change of emission intensity. For example, the first threshold value can be the slope +/- (plus or minus) up to <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%.

The slope satsifies the condition given by: <MAT>.

In one embodiment, when exciting the first detection moiety (and thereby obtaining a first image), a wavelength of a first excitation light can be selected to not excite the second detection moiety. Then, a wavelength of a second excitation light can also be selected to excite the first detection moiety (thereby providing a second image) and to not excite the second detection moiety. The first and second images are processed and compared to obtain the change of emission intensity (in other words, the slope, or y/x) based on the emissions of the first detection moiety due to the change in excitation wavelengths.

In one embodiment, at least one of the excitation lights can stimulate one or more detection moieties. However, the resulting slopes, as discussed below, can be used to remove the signal of one or more non-desired detection moieties.

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

In one embodiment, any of the methods or systems can be used for a plurality of stains on or within a sample or fraction thereof. The steps can be performed simulatenously for at least two of the stains or can be performed for a first stain and then for a second stain. In one embodiment, the first- or higher-order derivative can be calculated for each detection moiety spectral edge. In one embodiment, the spectral edge of the respective detection moieties can be used to differentiate between the emissions of the different detection moieties.

In one embodiment, the minimum number of raw images is n, where n is the number of detection moieties. For example, a first raw image can be obtained at a higher end of a trailing edge of a first emission spectrum and at 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 at 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 moiety (as depicted by the first emission spectrum) and a second final image of a second detection moiety (as depicted by the second emission spectrum). Though the emission spectra is discussed, this embodiment can be implemented on excitation spectra.

In one embodiment, the minimum number of raw images is n+<NUM>, where n is the number of detection moieties.

In one embodiment, all of the raw and final images of the first and second detection moieties are displayed to an end user or operator, such as on a screen (e.g., the screen of at least one of a phone, a tablet, a computer, a television, a PDA, a handheld device, or the like). In one embodiment, at least one of the raw images of the first and/or second detection moieties is displayed. In one embodiment, at least one of the final images of the first and/or second detection moieties is displayed. In one embodiment, none of the raw images are displayed but at least one of the final images is displayed. In one embodiment, none of the raw images are displayed but all of the final images are displayed.

The embodiments for acquiring signals (for example, 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 spectrum specifically discussed for the example acquisition. Rather, the signal acquisition can apply to one or more emission spectra, wherein all emission spectra have the same acquisition (for example, 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.

To obtain the raw images, the imaging can be done with a flow cytometer or a microscope, such as a fluorescent microscope, a scanner, or the like. Imaging can be done in, conventional epifluorescence, light sheet microscopy, super resolution microscopy, and confocal microscopy. <FIG> shows an optical path of a fluorescent microscope. The optical path includes an excitation source <NUM> which emits an excitation light <NUM>, such as a light in the visible, infrared ("IR"), or ultraviolet ("UV") spectra. The excitation light <NUM> comprises a plurality of wavelengths, including at least a first excitation wavelength <NUM> and a second excitation wavelength <NUM>. The excitation light <NUM> interacts with an excitation spectrum selector <NUM>, such that the first excitation wavelength <NUM> passes through the excitation spectrum selector <NUM> and the second excitation wavelength <NUM> is blocked from passing through the excitation spectrum selector <NUM>. The first excitation wavelength <NUM> is then reflected off a second filter <NUM>. The second filter <NUM> re-directs the first excitation light <NUM> into an objective <NUM>.

The objective <NUM> receives the first excitation wavelength <NUM> and focuses the first excitation wavelength at a point or surface on, within, or near a sample or fraction thereof <NUM>. The first excitation wavelength <NUM> stimulates a first detection moiety (not shown) on or with the sample or fraction thereof <NUM>, thereby causing the first detection moiety (not shown) to emit a first emission light <NUM> having a first emission wavelength. The first emission light <NUM> can be captured by the objective <NUM>, passed back through the second filter <NUM>, passed through an emission spectrum selector <NUM>, and onto an emission detector <NUM>. The emission detector <NUM> can be a charge-coupled device ("CCD"), CMOS camera, a scientific CMOS camera, photodiode, photomultiplier tube, or the like for capturing image data, which can then be compiled into images, processed and analyzed by a computer or associated software or programs.

The excitation source <NUM> emits the excitation light <NUM> again. The excitation light <NUM>, however, now interacts with the excitation spectrum selector <NUM>, such that the second excitation wavelength <NUM> passes through the excitation spectrum selector <NUM> and the first excitation wavelength <NUM> is blocked from passing through the excitation spectrum selector <NUM>. The second excitation wavelength <NUM> is then reflected off the second filter <NUM>. The second filter <NUM> re-directs the second excitation light <NUM> into the objective <NUM>. The objective <NUM> receives the second excitation wavelength <NUM> and focuses the first excitation wavelength at a point or surface on, within, or near a sample or fraction thereof <NUM>. The second excitation wavelength <NUM> stimulates the first detection moiety (not shown) on or with the sample or fraction thereof <NUM>, thereby causing the first detection moiety (not shown) to emit a second emission light <NUM> having a second emission wavelength. The second emission light <NUM> can be captured by the objective <NUM>, passed back through the second filter <NUM>, passed through the emission spectrum selector <NUM>, and onto an emission detector <NUM>.

The process discussed can be performed any number of times for any number of detection moieties.

The second filter <NUM> can each be a dichroic, polychroic, bandpass, bandstop, or any appropriate filter.

The sample or fraction thereof <NUM> can be located on a base <NUM> or between a cover <NUM> and the base <NUM>. The cover <NUM> and the base <NUM> can be optically clear to permit imaging. The sample <NUM>, the cover <NUM>, and the base <NUM> can be located on a platform <NUM> to move the sample <NUM> in an x-, y-, or z-direction as required. The platform <NUM> can include an aperture <NUM> which allows the first excitation wavelength <NUM>, having been focused by the objective <NUM>, into, on, or near the sample or fraction thereof <NUM>. The platform <NUM> can be driven by a driver <NUM>, which includes at least one of a z-direction drive <NUM>, an x-direction drive <NUM>, and a y-direction drive <NUM> to position the sample <NUM>. The driver <NUM> can be a motor, such as a servomotor or a stepper motor, a piezo-electric actuator, a solenoid, or the like.

The optical path can also include a cut-off aperture (not shown), such as in a confocal microscope, to increase the signal/noise ratio of the boundary light signal.

The base <NUM> can be composed of glass; an inert metal; a metal; a metalloid; organic or inorganic materials, and plastic materials, such as a polymer; and combinations thereof. The cover <NUM> can be composed of an optically transparent material.

<FIG> shows an optical path of a fluorescent microscope similar to that of <FIG>, except that the excitation source <NUM> emits the first and second excitation wavelengths as separate lights <NUM>, <NUM>.

<FIG> shows an optical path of a fluorescent microscope similar to that of <FIG>, except that the excitation spectrum selector <NUM> is not incorporated into the optical path.

In one embodiment, the excitation spectrum selector <NUM> or the emission spectrum selector <NUM> can be a filter to block or pass given wavelengths. In one embodiment, the excitation spectrum selector <NUM> or the emission spectrum selector <NUM> can be a notch filter, a bandstop filter, or a bandpass filter. In one embodiment, the excitation spectrum selector <NUM> or the emission spectrum selector <NUM> can be an acousto optic tunable filter or a liquid crystal tunable filter. In one embodiment, the excitation spectrum selector <NUM> or the emission spectrum selector <NUM> can be a diffraction grating. In one embodiment, the excitation spectrum selector <NUM> or the emission spectrum selector <NUM> can include a filter capable of being re-angled to block or pass given wavelengths based on the angle at which the filter is tilted, turned, or adjusted relative to an incoming light, thereby changing the angle of incidence of light upon the filter. As an example, the first excitation wavelength <NUM> passes through the excitation spectrum selector <NUM> and the second excitation wavelength <NUM> is blocked from passing through the excitation spectrum selector <NUM> due to the angle of the excitation spectrum selector. Then, the excitation spectrum selector <NUM> can be re-angled (θ) to block the first excitation wavelength <NUM> and pass the second excitation wavelength <NUM>. Though the example discusses the excitation spectrum selector <NUM>, the first excitation wavelength <NUM>, and the second excitation wavelength <NUM>, the optical pathway is not intended to be so limiting.

Re-angling can be applied to the emission spectrum selector <NUM> to block and pass certain emission wavelengths. Additionally, rotating or angling can be performed before or after capturing any raw image. In one embodiment, the emission spectrum selector <NUM> can include a first emission filter <NUM> capable of being rotated or angled from one angle of incidence (denoted by the longer dashed line filter <NUM>) between the first filter <NUM> and the emission light to another angle of incidence (denoted by the shorter dashed line filter <NUM>) between the first filter <NUM> and the emission light, including every angle in between. For example, a first raw image can be obtained with the first filter <NUM> at a first angle of incidence. Then the first filter <NUM> can be re-angled from the first angle of incidence to a second angle of incidence. A second raw image can then be obtained. Additionally, in capturing at least one more raw image, the first filter <NUM> can be re-angled from the second angle of incidence to a third angle of incidence. A third raw image can then be obtained. Additionally, in capturing at least one more raw image, the first filter <NUM> can be re-angled from the third angle of incidence to a fourth angle of incidence. A fourth raw image can then be obtained. In one embodiment, at least two of the first, second, third, and fourth angles are the same. In one embodiment, none of the first, second, third, and fourth angles are the same.

In one embodiment, the emission spectrum selector <NUM> can include two or more emission filters <NUM>, <NUM> such that each one is capable of being rotated or angled. The first filter <NUM> can be rotated or angled from one angle of incidence (denoted by the longer dashed line filter <NUM>) between the first filter <NUM> and the emission light to another angle of incidence (denoted by the shorter dashed line filter <NUM>, <NUM>) between the first filter <NUM> and the emission light, including every angle in between. The second filter <NUM> can be rotated or angled from a one angle of incidence (denoted by the longer dashed line filter <NUM>) between the second filter <NUM> and the emission light to another angle of incidence (denoted by the shorter dashed line filter <NUM>) between the second filter <NUM> and the emission light, including every angle in between. The first and second filters <NUM>, <NUM> can be rotated or angled independently of each other. In other words, the first filter <NUM> can have any number of positions (i.e., first position, second position, third position, fourth position, and so on until the nth position) with each position corresponding to a different angle. The second filter <NUM> can have any number of positions (i.e., first position, second position, third position, fourth position, and so on until the nth position) with each position corresponding to a different angle. Each filter <NUM>, <NUM> can have a position (or angle) independent of the other filter, such that one or both filters <NUM>, <NUM> can be angled or rotated, and such that the filters <NUM>, <NUM> can have the same angle of incident or difference angles of incidence. The filters <NUM>, <NUM> can be positioned or angled to obtain desired wavelengths.

For example, a first raw image can be obtained with the first filter <NUM> at a first angle of incidence and the second filter <NUM> at a third angle of incidence. Then the first filter <NUM> can be re-angled from the first angle of incidence to a second angle of incidence, while the second filter <NUM> stays at the third angle of incidence. A second raw image can then be obtained. Additionally, in capturing at least one more raw image, the second filter <NUM> can be re-angled from the third angle of incidence to a fourth angle of incidence. A third raw image can then be obtained. In one embodiment, at least two of the first, second, third, and fourth angles are the same. In one embodiment, none of the first, second, third, and fourth angles are the same.

In any of the embodiments including rotating or angling at least one filter, any filter can be rotated or angled at any desired time or step to obtain a desired emission wavelength. For example, after obtaining a first raw image with the first and second filters <NUM>, <NUM> at first and third angles of incidence, respectively, the first and second filters <NUM>, <NUM> can be rotated or angled to second and fourth angles of incidence, respectively. A second raw image can then be obtained. Then, one or both of the filters <NUM>, <NUM> can be rotated or angled. A third raw image can be obtained. In other words, each filter can be rotated or angled independently of the other filter or filters at any point and by any amount to obtain any raw image and/or any desired emission wavelength.

In one embodiment, a plurality of filters can be used, wherein the filters have narrow bandpass (for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>; <NUM>-<NUM>; or the like). Therefore, in addition to or instead of rotating, the filters can be changed in and out to provide desired emission or excitation wavelengths.

Furthermore, though one and two filters are discussed, any number of filters can be used, including, without limitation, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

Though the examples and embodiments discussed herein are applied to the emission spectrum selector <NUM>, one or more filters capable of being rotated or angled, and the methods of using the same, can also be applied to the excitation spectrum selector <NUM>. Accordingly, in one embodiment, at least one of the excitation and emission spectrum selectors <NUM>, <NUM> can include at least one filter capable of being rotated or angled. In one embodiment, one of the excitation and emission spectrum selectors <NUM>, <NUM> can include at least one filter capable of being rotated or angled. In one embodiment, both of the excitation and emission spectrum selectors <NUM>, <NUM> can include at least one filter capable of being rotated or angled.

The individual filters of the excitation spectrum selector <NUM> or the emission spectrum selector <NUM> or the angle of incidence between the filters and the excitation or emission light can be selected to the desired raw images at the lower and higher edges of the desired spectral edges. For example, in one embodiment, the detection moieties can have differences in spectra at their peaks of less than or equal to <NUM>. In one embodiment the detection moieties can have differences in spectra at their peaks of less than or equal to <NUM>. In one embodiment, the detection moieties can have differences in spectra at their peaks of <NUM>-<NUM>. In one embodiment, the detection moieties can have differences in spectra at their peaks of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In one embodiment, the difference between successive spectra (such as at the peak) can be the same (e.g., first and second detection moieties are separated by <NUM> and second and third detection moieties are separated by <NUM>). In one embodiment, the differences between successive spectra (such as at the peak) can be different (e.g., first and second detection moieties are separated by <NUM> and second and third detection moieties are separated by <NUM>).

In one embodiment, the angle of incidence of light upon any filter can be <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, <NUM>°, or <NUM>°. In one embodiment, the angle of incidence of light upon any filter can be up to, but not inclusive of, <NUM>°. In one embodiment, the angle of incidence of light upon any filter can be less than <NUM>°. In one embodiment, the angle of incidence of light upon any filter can be from <NUM>° to <NUM>°. As noted above, when there are two or more filters, each filter rotates freely and 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 angles of incidence are selected based on the desired wavelength to be obtained.

Any of the images or files, whether raw or processed, can be stored in any appropriate storage medium at any point during the performance of any embodiment of the present invention. The storage medium includes, but is not limited to, one or more of a hard disk, a random-access memory (RAM), a read only memory (ROM), a storage of distributed computing systems, an optical disk (such as a compact disc, digital versatile disc, or Blu-ray Disc), a flash memory device, a memory card, or the like.

Embodiments of the invention include a non-transitory computer readable medium which can store instructions for performing the above-described methods and any steps thereof, including any combinations of the same. For example, the non-transitory computer readable medium can store instructions for execution by one or more processors or similar devices.

Embodiments of the invention include two or more non-transitory computer readable media which can store instructions for performing the above-described methods and any steps thereof, including any combinations of the same. For example, the instructions for execution can be split amongst two or more processors or similar devices.

Further embodiments of the present invention can also include a computer or apparatus (e.g. a phone, a tablet, a PDA, or the like) which reads out and executes computer executable instructions, such as a non-transitory computer-readable medium, recorded or stored on a storage medium (which may be the same as or different than the storage medium for storing images or files, as discussed above), to perform the functions of any embodiment. The computer may include one or more of a central processing unit (CPU), micro processing unit (MPU), or other circuitry, and may include a network of separate computers or separate computer processors.

The computer or apparatus can also be configured to display, such as on a monitor or screen, any of the images or files, whether raw or processed.

A sample labeled with Sytox Orange and CF <NUM> is provided and imaged. <FIG> shows a first raw image at emisson wavelength <NUM> (+/- <NUM>). <FIG> shows a second raw image at emission wavelength <NUM> (+/- <NUM>). <FIG> shows a third raw image at emission wavelength <NUM> (+/- <NUM>). One or more signals within the first, second, and third raw images are provided by one or more of autofluorescence, Sytox Orange, and CF <NUM>. <FIG> shows a final image of Sytox Orange after the first, second, and third raw images undergo analysis and processing based on emission curvatures, as discussed above, to at least partially, if not completely, remove signals caused by autofluorescence and CF <NUM>.

The sample labeled with Sytox Orange and CF <NUM> is provided and imaged. <FIG> shows the second raw image at emission wavelength <NUM> (+/- <NUM>). <FIG> shows a fourth raw image at emission wavelength <NUM> (+/- <NUM>). One or more signals within the second and fourth raw images are provided by one or more of autofluorescence, Sytox Orange, and CF <NUM>. <FIG> shows a final image of CF <NUM> after at least two of the first, second, third, and fourth raw images undergo analysis and processing based on emission slopes, as discussed above, to at least partially, if not completely, remove signals caused by autofluorescence and Sytox Orange.

<FIG> shows a final image of the autofluorescence after at least two of the first, second, third, and fourth raw images undergo analysis and processing based on emission curvatures, as discussed above, to at least partially, if not completely, remove signals caused by the Sytox Orange and CF <NUM>. In other words, <FIG> depicts the autofluorescence of a particular structure of the sample.

Spatially, graphically, or numerically relative terms, such as "under", "below", "lower", "over", "upper", "higher", and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations when in use or operation in addition to the orientation depicted in the figures. For example, if a device, system, or method, as depicted in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Additionally, "lower", "higher", and the like are used to depict elements, features, information, or the like which, relative to each other or at least other elements, features, information, or the like are further down or further up a chart, graph, or plot, or are lesser or greater in value or intensity.

Additionally, though "first" and "second" are used, the terms are not intended to limit various features/elements to only one or two. Rather, three (i.e., third), four (i.e., fourth), or more may be included or used where appropriate or desirous to do so.

The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of the invention as it is set forth in the claims. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term "invention" merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claim 1:
A method of analyzing a sample, the sample comprising a first detection moiety having a first emission spectrum (<NUM>), the first emission spectrum having a peak emission (<NUM>) intensity at a peak intensity wavelength, a leading spectral edge (<NUM>) with emission intensities less than the peak intensity at wavelengths less than the peak intensity wavelength, and a trailing spectral edge (<NUM>) with emission intensities less than the peak intensity at wavelengths greater than the peak intensity wavelength, the method comprising:
acquiring, with an imaging device by stimulating the sample with excitation light, a first set of raw images, the first set of raw images consisting of:
a first raw image comprising a first emission signal while stimulating the sample with the excitation light, wherein the first emission signal comprises an intensity at a first emission wavelength of the first detection moiety, and
a second raw image comprising a second emission signal while stimulating the sample with the excitation light, wherein the second emission signal comprises an intensity at a second emission wavelength of the first detection moiety,
wherein the intensity of the first emission signal is less than the intensity of the second emission signal within the first emission spectrum;
calculating a slope of a percentage change in signal intensity between pixels of the first raw image and the corresponding pixels of the second raw image, the slope between the respective pixels defined as the percentage change between the intensity of the first emission signal and the intensity of the second emission signal divided by the change in the first emission wavelength and the second emission wavelength;
wherein if the calculated slope is at least plus or minus (+/-) <NUM>%/nm, then the pixels of the first raw image and the pixels of the second raw image are attributed to the first detection moiety; and
outputting, with a processor, a final image of the first detection moiety based on the first and second raw images.