WO2025059475A1 - Methods, systems, and devices for using autofluorescence to probe cellular characteristics - Google Patents

Abstract

Disclosed herein are methods, systems, and devices for probing cellular characteristics. Steps can include preparing a biological specimen for a spectral analysis and exciting the biological specimen by at least one excitation wavelength. Steps can further include creating an emission spectrum related to the excitation wavelength by detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum. The biological specimen includes at least one analytical target. Steps can further include collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum and establishing an AF spectrum related to the analytical target. The AF spectrum can be used to probe at least one cellular characteristic.

Description

METHODS, SYSTEMS, AND DEVICES FOR USING AUTOFLUORESCENCE TO PROBE CELLULAR CHARACTERISTICS

FIELD

[0001] This disclosure relates to a method of using autofluorescence as a proxy for a cellular characteristic, particularly when using a spectral analysis such as, but not limited to, spectral flow cytometry and/or spectral fluorescence microscopy.

BACKGROUND

[0002] Conventional flow cytometry is a methodology that enables detection and analysis of cells suspended in a buffered salt-based solution as they flow hydrodynamically along a focal plane through single/multiple lasers. A laser can excite intrinsic fluorescent properties both on the surface of cells and intracellularly. Light emitted by these properties is collected at fine resolution wavelengths by optical detectors and is converted digitally to light intensity values in units referred to as relative fluorescence units (RFUs). Emission data points can be collected from many tens to hundreds of detectors. After labelling the cells with fluorophore-conjugated antibodies or dyes that binds to specific cellular components, different cell types can be identified and distinguished from each other.

[0003] Conventional fluorescence microscopy is used in many fields of biology to visualize cellular structures and study protein localization. Fluorescence microscopy utilizes a laser to excite fluorophores in a biological sample. The biological sample is pre-labeled with a fluorophore that attaches (for example, a fluorescent antibody) to an analytical target, such as a protein or other cellular component of interest. The sample is then placed on a slide and put under the lens of the microscope. The laser excites the fluorophore at an excitation wavelength, which then produces an emission signal at a different wavelength. The emitted signal is captured by a photodetector. The fluorescence microscope can therefore show the spatial distribution of the analytical target within the biological sample.

[0004] In conventional flow cytometry and fluorescence microscopy, optical filters allow the collection of a portion of the emitted light (usually, around the peak wavelength) by the photodetectors. Autofluorescence (AF) is the ability of a molecule to absorb and emit a spectrum of light. AF in cells and body tissues occurs due to the absorbance of light by specific substances in cellular membrane, cytoplasm, intracellular organelles and/or extracellular contents. Because of AF’s wide emission wavelengths, low intensity, and lack of means to capture its whole spectrum, it is considered as noise signal in conventional flow cytometry and microscopy (1, 2, 7).

[0005] These conventional tools are still limited and can be improved by the methods, systems, and devices disclosed herein.

SUMMARY

[0006] In one aspect, disclosed herein is a method of probing a cellular characteristic, the method comprising preparing a biological specimen for a spectral analysis, the biological specimen comprising at least one analytical target, exciting the biological specimen with at least one excitation laser, the excitation laser having an excitation wavelength, creating an emission spectrum related to the excitation wavelength by detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum, collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum, establishing an AF spectrum related to the analytical target using the autofluorescence measurements from across the emission spectrum, and using the AF spectrum to probe a cell age.

[0007] In one aspect, disclosed herein is a method of probing a cellular characteristic, the method comprising preparing a biological specimen for a spectral analysis, the biological specimen comprising at least one analytical target, exciting the biological specimen with at least one excitation laser, the excitation laser having an excitation wavelength, creating an emission spectrum related to the excitation wavelength by detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum, collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum, establishing an AF spectrum related to the analytical target using the autofluorescence measurements from across the emission spectrum, and using the AF spectrum to probe a cell type.

[0008] In some implementations, the at least one excitation laser comprises a plurality of excitation lasers, each excitation laser of the plurality of excitation lasers having a differing excitation wavelength than the other excitation lasers of the plurality of excitation lasers. In some implementations, the method further comprises creating a plurality of emission spectrums, each emission spectrum of the plurality of emission spectrums related to a particular excitation wavelength from the plurality of excitation lasers. In some implementations, the method further comprises sequentially stimulating the biological specimen by exciting the biological specimen with a first excitation laser of the plurality of excitation lasers, pausing from 15 to 65 microseconds, then exciting the biological specimen with a next excitation laser of the plurality of excitation lasers, thereby creating a sequential stimulation.

[0009] In some implementations, the total duration of the sequential stimulation is from 25 to 600 microseconds. In some implementations, detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum comprises detecting emission in at least two of the ultraviolet, visible, and infrared ranges.

[0010] In some implementations, the biological specimen comprises a cell suspension. In some implementations, the biological specimen comprises a tissue sample. In some implementations, the biological specimen comprises a bodily fluid. In some implementations, the method comprises preparing the biological specimen comprises preparing a slide. In some implementations, the method comprises preparing the biological specimen comprises preparing a cell suspension for a spectral cytometry assay.

[0011] In some implementations, the analytical target is a cell population. In some implementations, the cell population is a mammalian cell population. In some implementations, the analytical target is a cellular component. In some implementations, the cellular component comprises a membrane, an organelle, or an extracellular matrix material.

[0012] In some implementations, the method comprises collecting AF measurements from the analytical target comprising identifying emission signals from the analytical target. In some implementations, the method comprises establishing an AF spectrum related to the analytical target comprising applying a spectral unmixing algorithm. In some implementations, the method comprises using the AF spectrum to probe a cellular characteristic comprising comparing the AF spectrum related to the analytical target to at least one control AF value.

[0013] In some implementations, the at least one control AF value is from a different biological specimen with known properties. In some implementations, the control AF value is predetermined.

[0014] In some implementations, the cellular characteristic is a cell type. In some implementations the cellular characteristic is a cell age. In some implementations, the cellular characteristic is a cell health. In some implementations, the cellular characteristic is a response to a treatment.

[0015] In some implementations, the method is performed without applying a cellular stain to the biological specimen. In some implementations, the spectral analysis utilizes spectral cytometry. In some implementations, the spectral analysis utilizes spectral fluorescence microscopy.

BRIEF DESCRIPTION OF FIGURES

[0016] Figure 1 shows a quantitative analysis of age-related AF changes in primary mouse microglial cells. Samples went through standard brain tissue dissociation into single cell suspension and Percoll gradient to eliminate myelin debris. Enriched cells were stained with CDllb-BV431 and CD45-APC, washed with FACS buffer before acquiring spectral data on ID7000 (UC Davis) with six excitation lasers. Microglia were gated as CDllb+CD451ow. Data come from three independent experiments on male mice.

[0017] Figure 2A shows a quantitative analysis of age-related AF changes in different primary mouse brain cells. Samples went through standard brain tissue dissociation into single cell suspension and Percoll gradient to eliminate myelin debris. Enriched cells were stained with CDl lb-BV431 and CD45-APC, washed with FACS buffer before acquiring spectral data on ID7000 (UC Davis) with six excitation lasers. Microglia were gated as CDllb+CD451ow, macrophages as CDl lb+CD45high, other immune cells as CD11-CD45+, neurons and glia as CD1 lb-CD45-. Data come from three independent experiments on male mice. Figure 2B shows the gating strategy using CD45-APC and CDl lb-BV421 to identify microglia

(CD1 lb+CD451ow), peripherally-derived macrophages (CD1 lb+CD45high), other immune cells (CDllb-CD45high) and Neurons/glia cells (CD1 lb-CD45+). The data is from a ID7000 spectral analyzer with seven excitation lasers.

[0018] Figures 3A and 3B show a quantitative analysis of age-related AF changes in primary mouse brain CDl lb+ cells. Sample went through standard brain tissue dissociation into single cell suspension and Percoll gradient to eliminate myelin debris. Enriched cells were stained with CD1 lb-APC-eF780, washed with FACS buffer before acquiring spectral data on BD FACSymphony A3 (Altos Labs). Figure 3A shows a relative AF spectrum normalized on 3mo. mice. Figure 3B shows an area under the curve quantification. Data come from three independent experiments on male mice.

[0019] Figure 4 shows a quantitative analysis of age-related AF changes in primary mouse microglial cells. Samples went through standard brain tissue dissociation into single cell suspension and Percoll gradient to eliminate myelin debris. Enriched cells were stained with CD1 lb-BV431 and CD45-APC, washed with FACS buffer before acquiring spectral data on ID7000 (UC Davis) with six excitation lasers. Microglia were gated as CDllb+CD451ow. Data come from three independent experiments on female mice. [0020] Figure 5A shows a quantitative analysis of age-related AF changes in different primary mouse brain cells. Samples went through standard brain tissue dissociation into single cell suspension and Percoll gradient to eliminate myelin debris. Enriched cells were stained with CD1 lb-BV431 and CD45-APC, washed with FACS buffer before acquiring spectral data on ID7000 (UC Davis) with six excitation lasers.. Microglia were gated as CDl lb+CD451ow, macrophages as CD1 lb+CD45high, other immune cells as CD1 1 -CD45+, neurons and glia as CDl lb-CD45-. Data come from three independent experiments on female mice. Figure 5B shows the gating strategy using CD45-APC and CDllb-BV421 to identify microglia (CDllb+CD451ow), peripherally-derived macrophages (CDl lb+CD45high), other immune cells (CD1 lb-CD45+) and Neurons/glia cells (CD1 lb-CD45-).

[0021] Figures 6A and 6B shows a quantitative analysis of age-related AF changes in primary mouse brain CDl lb-i- cells. Sample went through standard brain tissue dissociation into single cell suspension and Percoll gradient to eliminate myelin debris. Enriched cells were stained with CDl lb-APC-eF780, washed with FACS buffer before acquiring spectral data on BD FACSymphony A3 (Altos Labs). Figure 6A shows a relative AF spectrum normalized on 3mo. mice. Figure 6B shows an area under the curve quantification. Data come from three independent experiments on female mice.

[0022] Figures 7A, 7B, and 7C show the microglia specific autofluorescence in young (3 mo) vs. old (21 mo) mouse brain slices. The data presented in Figure 7A, 7B, and 7C are from a different ID7000 spectral analyzer having an additional excitation laser (seven total excitation lasers). Figure 7A shows the mean fluorescence intensity (MFI) in the microglia of 3 mo. vs. 21 mo. mice. Figure 7B shows the MFI in the microglia of 3 mo. vs. 21 mo. male mice. Figure 7C shows the MFI in the microglia of 3 mo. vs. 21 mo. female mice.

[0023] Figures 8A, 8B, and 8C show the macrophage specific autofluorescence. Figure 8A shows the mean fluorescence intensity (MFI) in the macrophages of 3 mo. vs. 21 mo. mice. Figure 8B shows the MFI in the macrophages of 3 mo. vs. 21 mo. male mice. Figure 8C shows the MFI in the macrophages of 3 mo. vs. 21 mo. female mice.

[0024] Figures 9A, 9B, and 9C show the specific autofluorescence in other immune cells. Figure 9A shows the mean fluorescence intensity (MFI) in the other immune cells of 3 mo. vs. 21 mo. mice. Figure 9B shows the MFI in the other immune cells of 3 mo. vs. 21 mo. male mice. Figure 9C shows the MFI in the other immune cells of 3 mo. vs. 21 mo. female mice.

[0025] Figures 10A, 10B, and 10C show the neuron and glia specific autofluorescence (MFI are noted to be low indicating presence of artifacts). Figure 10A shows the mean fluorescence intensity (MFI) in the neuron and glia of 3 mo. vs. 21 mo. mice. Figure 10B shows the MFI in the neuron and glia of 3 mo. vs. 21 mo. male mice. Figure IOC shows the MFI in the neuron and glia of 3 mo. vs. 21 mo. female mice.

[0026] Figure 11 shows the microglia specific autofluorescence in young (5mo) vs. old (20 mo) mouse brain slices using an AiryScan 980 Spectral Scanner.

DETAILED DESCRIPTION

[0027] The following description of certain examples of the inventive concepts should not be used to limit the scope of the claims. Other examples, features, aspects, configurations, embodiments, and advantages will become apparent to those skilled in the art from the following description. As will be realized, the device and/or methods are capable of other different and obvious aspects, all without departing from the spirit of the inventive concepts. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive.

[0028] For purposes of this description, certain advantages and novel features of the aspects and configurations of this disclosure are described herein. The described methods, systems, and apparatus should not be construed as limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and aspects of the various disclosed aspects, alone and in various combinations and sub-combinations with one another. The disclosed methods, systems, and apparatus are not limited to any specific aspect, feature, or combination thereof, nor do the disclosed methods, systems, and apparatus require that any one or more specific advantages be present or problems be solved.

[0029] Although the operations of exemplary aspects of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that disclosed aspects can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular aspect or implementation are not limited to that aspect or implementation, and may be applied to any aspect or implementation disclosed. It will understood that various changes and additional variations may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention or the inventive concept thereof. Certain aspects and features of any given aspect may be translated to other aspects described herein. In addition, many modifications may be made to adapt a particular situation or device to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular implementations disclosed herein, but that the invention will include all implementations falling within the scope of the appended claims.

[0030] Features, integers, characteristics, compounds, chemical moieties, or groups described in conjunction with a particular aspect, configuration, embodiment or example of the invention are to be understood to be applicable to any other aspect, configuration, embodiment, or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing aspects. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract, and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0031] Throughout this application, various publications and patent applications may be referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains. However, it should be appreciated that any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material. [0032] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non- limiting aspect the terms are defined to be within 10%. In another non-limiting aspect, the terms are defined to be within 5%. In still another non-limiting aspect, the terms are defined to be within 1%.

[0033] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0034] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. "Exemplary" means "an example of" and is not intended to convey an indication of a preferred or ideal aspect. "Such as" is not used in a restrictive sense, but for explanatory purposes.

[0035] Excitation and emission refer to the processes by which fluorophores absorb and emit light. When a fluorophore is exposed to light at a specific excitation wavelength, it absorbs the energy and its electrons are promoted to a higher energy state. After a brief period, the electrons return to their ground state, releasing energy in the form of light at a longer, lower-energy emission wavelength. The difference between the excitation and emission wavelengths is known as the Stokes shift, and this shift allows the emitted light to be distinguished from the excitation light, enabling fluorescent imaging or analysis.

[0036] Fluorophores are compounds or molecules that luminesce and/or emit light. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores encompassed by this disclosure include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy- X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- 1 methylcoumarin; 9-Amino-6-chloro-2- methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9

(Bis aminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; Blancophor FFG; Blancophor SV; BOBO™ - 1; BOBO™-3; Bodipy 492/515; Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca²⁺ Dye; Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green- C18 Ca²⁺; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF;

CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilCl 8(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilCl 8(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilCl 8(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo- 4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 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; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold);

Hydroxy tryptamine; 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 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; ; 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-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin;

Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO- 1; POPO-3; PO-PRO-1; PO- 1 PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl);

PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1;

Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6- methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C;

Sulphorhodamine 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; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO- 3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO- 1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.

[0037] Autofluorescence occurs when biological structures, such as proteins, lipids, and metabolites, naturally emit fluorescence without the need for external fluorophores. This phenomenon is driven by intrinsic fluorophores like NADH, collagen, and flavins, which absorb light and emit at specific wavelengths similar to synthetic fluorescent dyes. When excited by specific wavelengths, these molecules emit light in the visible spectrum, contributing to background signals in fluorescence microscopy or flow cytometry. While autofluorescence can complicate the interpretation of results by introducing unwanted background noise, it can also be exploited for imaging and analysis of specific tissue properties, especially in metabolic studies or label-free imaging techniques.

[0038] Autofluorescing cellular components can include (but are not limited to) NADH, FAD (Flavin Adenine Dinucleotide), collagen, elastin, lipofuscin, riboflavin, FMN (Flavin Mononucleotide), porphyrins, tryptophan, tyrosine, phenylalanine, retinol (Vitamin A), retinal, retinoic acid, bilirubin, chlorophyll, lipoproteins, cholesterol, cholesterol derivatives, melanin, serotonin, ubiquinone (Coenzyme Q), pyridoxine (Vitamin B6), and advanced glycation end products (AGEs). Examples of advanced glycation end products (AGEs) include carboxymethyllysine (CML), pentosidine, glucosepane, methylglyoxal-derived hydroimidazolone (MG-H1), glyoxal-lysine dimer (GOLD), and pyrraline.

[0039] The disclosure provides methods that are useful for applications including, but not limited to disease diagnosis, disease prevention, and/or disease treatment. Non-limiting examples of diseases include neurodegenerative diseases, proliferative diseases (such as, for example cancers), infectious diseases, congenital diseases, and metabolic diseases. [0040] In some embodiments, the neurodegenerative disease includes, but is not limited to Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, spinal muscular atrophy, Alpers’ disease, Batten disease, Cerebro-oculo-facio-skeletal syndrome, Leigh syndrome, Prion diseases, monomelic amyotrophy, multiple system atrophy, striatonigral degeneration, motor neuron disease, multiple sclerosis (MS), Creutzfeldt-Jakob disease, Parkinsonism, spinocerebellar ataxia, dementia, and other related diseases.

[0041] In some embodiments, the cancer includes, but is not limited to brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), choriocarcinoma, chordoma, craniopharyngioma, hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), primitive neuroectodermal tumor (PNT), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), synovioma, or other forms of cancer.

[0042] In some embodiments, the infectious disease includes, but is not limited to common cold, influenza ( including, but not limited to human, bovine, avian, porcine, and simian strains of influenza), measles, acquired immune deficiency syndrome/human immunodeficiency virus (AIDS/HIV), anthrax, botulism, cholera, Campylobacter infections, chickenpox, chlamydia infections, cryptosporidosis, dengue fever, diphtheria, hemorrhagic fevers, Escherichia coli (E. coli) infections, ehrlichiosis, gonorrhea, hand-foot- mouth disease, hepatitis A, hepatitis B, hepatitis C, legionellosis, leprosy, leptospirosis, listeriosis, malaria, meningitis, meningococcal disease, mumps, pertussis, polio, pneumococcal disease, paralytic shellfish poisoning, rabies, rocky mountain spotted fever, rubella, salmonella, shigellosis, small pox, syphilis, tetanus, trichinosis (trichinellosis), tuberculosis (TB), typhoid fever, typhus, west nile virus, yellow fever, yersiniosis, zika, or other infectious diseases.

[0043] In some embodiments, the congenital disease includes, but is not limited to albinism, amniotic band syndrome, anencephaly, Angelman syndrome, Barth syndrome, chromosomal abnormalities (including, but not limited to abnormalities to chromosome 9, 10, 16, 18, 20, 21, 22, X chromosome, and Y chromosome), cleft lip/palate, club foot, congenital adrenal hyperplasia, congenital hyperinsulinism, congenital sucrase-isomaltase deficiency (CSID), cystic fibrosis, De Lange syndrome, fetal alcohol syndrome, first arch syndrome, gestational diabetes, Haemophilia, heterochromia, Jacobsen syndrome, Katz syndrome, Klinefelter syndrome, Kabuki syndrome, Kyphosis, Larsen syndrome, Laurence-Moon syndrome, macrocephaly, Marfan syndrome, microcephaly, Nager’s syndrome, neonatal jaundice, neurofibromatosis, Noonan syndrome, Pallister-Killian syndrome, Pierre Robin syndrome, Poland syndrome, Prader-Willi syndrome, Rett syndrome, sickle cell disease, Smith-Lemli-Optiz syndrome, spina bifida, congenital syphilis, teratoma, Treacher Collins syndrome, Turner syndrome, Umbilical hernia, Usher syndrome, Waardenburg syndrome, Werner syndrome, Wolf-Hirschhorn syndrome, Wolff-Parkinson- White syndrome, and other congenital diseases or disorders.

[0044] In some embodiments, the metabolic disease includes, but is not limited to diabetes mellitus Type I, diabetes mellitus Type II, familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe syndrome, metachromatic leukodystrophy, Niemann-Pick syndrome, phenylketonuria (PKU), Tay-Sachs disease, Wilson’s disease, hemachromatosis, mitochondrial disorders or diseases (including, but not limited to Alpers Disease; Barth syndrome; beta.- oxidation defects:camitine-acyl-carnitine deficiency; carnitine deficiency; coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency: Complex V deficiency; cytochrome c oxidase (COX) deficiency, LHON Leber Hereditary Optic Neuropathy; MM Mitochondrial Myopathy: LIMM Lethal Infantile Mitochondrial Myopathy; MMC Maternal Myopathy and Cardiomyopathy; NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease: FICP — Fatal Infantile Cardiomyopathy Plus, a MELAS- ssociated cardiomyopathy: MELAS Mitochondrial Encephalomyopathy with Lactic Acidosis and Strokelike episodes; LDYT Leber’s hereditary optic neuropathy and Dystonia; MERRF Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM Maternally inherited Hypertrophic CardioMyopathy; CPEO Chronic Progressive External Opthalmoplegia; KSS Kearns Sayre Syndrome; DM Diabetes Mellitus; DMDF Diabetes Mellitus+DeaFness; CIPO Chronic Intestinal Pseudoobstruction with myopathy and Opthalmoplegia; DEAF Maternally inherited DEAFness or aminoglycoside-induced DEAFness; PEM Progressive encephalopathy; SNHL SensoriNeural Hearing Loss; Encephalomyopathy; Mitochondrial cytopathy: Dilated Cardiomyopathy: GER Gastrointestinal Reflux: DEMCHO Dementia and Chorea; AMDF Ataxia, Myoclonus; Exercise Intolerance: ESOC Epilepsy, Strokes, Optic atrophy, & Cognitive decline; FBSN Familial Bilateral Striatal Necrosis: FSGS Focal Segmental Glomerulosclerosis: LIMM Lethal Infantile Mitochondrial Myopathy; MDM Myopathy and Diabetes Mellitus: MEPR Myoclonic Epilepsy and Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCM Maternally Inherited Hypertrophic CardioMyopathy; MICM Maternally Inherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardiomyopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION Nonarteritic Anterior Ischemic Optic Neuropathy; NIDDM Non-Insulin Dependent Diabetes Mellitus; PEM Progressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTT Rett Syndrome: SIDS Sudden Infant Death Syndrome: MIDD Maternally Inherited Diabetes and Deafness; and MODY Maturity-Onset Diabetes of the Young, and MNGIE), and other metabolic diseases.

[0045] A biological specimen can be, or can be derived from, a cell suspension, a tissue sample, and/or bodily fluid. For example, a biological specimen can be, or can be derived from, a tissue (e.g., tissue biopsy), an organ, a cell (including a cell maintained in culture), a cell lysate (or lysate fraction), or a biomolecule derived from a cell or cellular material (e.g. a polypeptide or nucleic acid). [0046] Non-limiting examples of body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid.

[0047] The methods, systems, and devices disclosed herein provide ways to probe cellular characteristics by establishing an autofluorescence (AF) spectrum for a particular analytical target. The probable cellular characteristics include, but are not limited to, a cell age, a cell health, a cell type, a cell state, a stage in a cell cycle, or a cellular response to a treatment. As we age, our cells accumulate genetic and epigenetic changes that ultimately lead to change of functionalities. Genetic mutations happen randomly which makes it almost impossible to use as a measurement of age. Age associated epigenetic changes (e.g. DNA methylation) occur in patterns, which has been used to develop epigenetic clocks to measure tissue and cellular ages. However, current epigenetic measurements are costly and time consuming, and cells being measured must be destroyed prior to DNA extraction. For example, a DNA methylation array on 10 samples would take a few weeks that involves cell/tissue lysis, DNA extraction and purification, library preparation, chip based array, data curation and following analyses. In contrast, the present disclosure provides a quick measurement of cellular age and functionality in multiple cell types. More importantly, the same cells being measured are kept alive and can be recovered for downstream cultures and further tests.

[0048] This disclosure encompasses spectral analyses of cellular AF as a biomarker to measure aging of cells, using spectral analyses of cellular AF as a proxy of cell aging for screening of rejuvenation drugs/reagents, and using spectral analyses of cellular AF as a proxy of cell activation/functionality for screening of drugs. Though the examples herein are in the context of nervous system cells, it should be understood that the methods, systems, and devices can be applied to biological specimens of any tissue type and the analytical targets can be, or can be derived from, any cell type of the mammalian body.

[0049] Spectral analyses are somewhat new in the context of biological imaging. Up until recently, cellular imaging (including of any cellular AF) utilized conventional flow cytometers and fluorescent light microscopies. Newer spectral analysis tools include, but are not limited to, spectral flow cytometry and spectral fluorescent microscopy. Spectral flow cytometry is an advanced variation of traditional flow cytometry that uses/detects the entire emission spectrum of fluorophores, rather than just detecting light in specific wavelength bands. Instead of traditional detectors that only measure light at certain predefined wavelengths, spectral flow cytometry uses multiple detectors (or a detector array) to capture the entire emission spectrum of each fluorophore across a broad range of wavelengths. This allows for more precise and nuanced detection of multiple markers on cells, even when their emission spectra overlap, leading to improved resolution and data collection from complex samples. Since many fluorophores have overlapping emission spectra, the system uses computational algorithms to "unmix" these spectra, attributing the appropriate portion of the signal to each fluorophore. This allows the detection of multiple fluorophores that would otherwise be indistinguishable in traditional flow cytometry. The system collects these spectral signatures for each cell and matches them to the specific fluorophores used. Data analysis software then interprets this data to identify and quantify the different cell populations based on the expression of specific markers. As disclosed and described herein, cellular AF can be analyzed as a separate parameter with enough specificity and sensitivity as if it were another fluorophore in the panel.

[0050] Spectral fluorescent microscopy builds on conventional fluorescence microscopy techniques but utilizes multiple excitation lasers and multiple detectors. Like spectral flow cytometry, spectral fluorescent microscopy uses the entire emission spectrum of fluorescent dyes (fluorophores) to enhance imaging capabilities. This technique improves the resolution and discrimination of fluorophores in microscopy, allowing for the visualization of multiple markers even if their emission spectra overlap. Unlike traditional fluorescence microscopy, which detects light in specific wavelength bands using filters, spectral fluorescence microscopy captures the entire emission spectrum of each fluorophore. A spectrometer or a detector array is used to collect this spectral data across a wide range of wavelengths. After spectral unmixing, the data is processed to generate high-resolution images that display the distribution of each fluorescent marker within the sample. These images can show multiple colors corresponding to different fluorophores, revealing detailed information about the spatial arrangement of the labeled molecules. As disclosed and described herein, cellular AF can be analyzed as a separate parameter with enough specificity and sensitivity as if it were another fluorophore in the panel. [0051] By utilizing spectral analyses to probe cellular characteristics via cellular AF patterns, the disclosed systems and methods provide quicker measurement and analysis at higher throughput as compared to existing technologies for measuring cellular aging/functionality. For example, use of spectral analyses to measure autofluorescence provides for quicker measurement than conventional imaging. With traditional fluorescent microscopy, it could take days to acquire spectral images of 10 samples. It then requires very specialized analyzing software (or codes in open-source software such as Fiji) and multiple hours, even days of computing and analyzing time. Using the disclosed systems and methods, data can be acquired at less than 1 minute per sample, and the analysis takes minutes for 10 samples using commercialized and widely used software, e.g. Flowjo. Furthermore, the disclosed systems and methods can be scaled up to measure hundreds of samples within a day, which is extremely hard to achieve using the DNA methylation arrays or spectral imaging.

[0052] The methods of probing a cellular characteristic disclosed herein include steps for preparing a biological specimen for a spectral analysis, such as (but not limited to) spectral cytometry or spectral fluorescence microscopy. The biological specimen can be, for example, a cell suspension, a tissue sample, and/or bodily fluid. For example, the biological specimen can comprise a sample of neurological tissue and/or a cell suspension derived from neurological tissue.

[0053] The preparation of the biological specimen can include, for example, fixing a sample, cryoprotecting a sample, embedding a sample for cryosectioning, cryosectioning a samples onto a slide, preparing a slide, blocking a slide, staining a slide, dissociating a tissue, preparing a cell suspension, enriching a specified cell population, applying a fluorophore or a cellular stain, or any steps required for the preparation of a sample for spectral analysis. Autofluorescence can be detected without use of fluorophores, but fluorophores can be added to distinguish between the cell types of a sample. That said, in some implementations, the methods might not include a step of applying a fluorophore or cellular stain to the biological specimen.

[0054] The biological specimen includes at least one analytical target from which AF measurements will be collected. In some implementations, the analytical target is a cell population (such as, but not limited to, a mammalian cell population). In some implementations, the cell population is a microglial cell, a neuron, a glial cell, a fibroblast (including, but not limited to: primary fibroblasts and/or cells from fibroblast cell lines), an adipocyte, a hepatocyte, a retinal pigment epithelium (RPE) cell (including, but not limited to: primary RPE cells and cells from RPE cell lines), a macrophage (including, but not limited to: macrophages from central nervous system (CNS) tissues/organs and/or macrophages from non-CNS tissues/organs), or another immune cell type. In some implementations, the analytical target is a cellular component such as, but not limited to, a membrane, an organelle, an extracellular matrix material, or a cellular molecule. In some implementations, the analytical target can be any auto fluorescing cellular component including (but not limited to) lipofuscin, NADH, FAD, advanced glycation end products, collagen, and elastin.

[0055] The methods of probing a cellular characteristic disclosed herein include steps for exciting the biological specimen with at least one excitation laser (the excitation laser having an excitation wavelength). Some implementations include a plurality of excitation lasers with a plurality of different excitation wavelengths. Some implementations may use 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more excitation lasers and, as such, may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more different excitation wavelengths.

[0056] The methods of probing a cellular characteristic further include creating an emission spectrum related to an excitation wavelength. An emission spectrum related to a particular excitation wavelength is created by detecting emission signals at a plurality of wavelengths across the electromagnetic (EM) spectrum (i.e., utilizing multiple detectors to detect wavelengths across the EM spectrum). The methods disclosed herein encompass the creation of a plurality of emission spectrums, each emission spectrum related to a particular excitation wavelength from the plurality of excitation lasers. In some implementations, emission is detected in at least two of the ultraviolet, visible, and infrared ranges. In some implementations, emission is detected in cell-type specific ranges. In some implementations, emission is detected by photomultiplier tubes (PMTs) and/or photodiodes (PDs) in cell-type specific combinations.

[0057] The methods disclosed herein further include collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum and establishing an AF spectrum related to the analytical target using the autofluorescence measurements from across the emission spectrum. AF signals from the analytical target are identified as being signals from the analytical target, and the AF spectrum is established by applying a spectral unmixing algorithm. The AF spectrum is then used to probe a cellular characteristic. This can be done by, for example, comparing the AF spectrum related to the analytical target to at least one control AF value or one control AF spectrum. This known AF control value or control AF spectrum can be predetermined and/or can be from a different biological specimen with known properties.

[0058] Some implementations include sequentially stimulating the biological specimen. Sequential stimulation is done by exciting the biological specimen with a first excitation laser, pausing from about 15 to about 65 microseconds (or about 25 to about 55 microseconds, or about 35 to about 45 microseconds), then exciting the biological specimen with a next excitation laser in an excitation laser series. This pause between excitation lasers allows the fluorophores to fully relax from one excitation wavelength before being excited by the next excitation wavelength. In some implementations, the total duration of the sequential stimulation is from about 25 to about 600 microseconds (or about 100 to about 550 microseconds, or about 200 to about 400 microseconds, or about 300 microseconds).

[0059] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. [0060] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

EXAMPLE 1

[0061] Background: Microglia are the immune resident cells of the central nervous system. It has been reported that AF of microglia increases with age due to the accumulation of lipids and misfolded proteins (3-6). In these reports, microglia AF was detected using conventional flow cytometry or fluorescence microscopy by measuring non-specific signal emitted after excitation with a single laser. These approaches provided limited data on the more complex AF spectrum of microglia cells.

[0062] For the following experiments, it was hypothesized that use of a spectral analyzer and/or a spectral cytometer could instead enable a comprehensive and specific exploration of autofluorescence in microglia cells, providing a tool for quantitative analysis of age-dependent or cell-type dependent changes in AF spectrums. The disclosure relates to a method for the measurement of AF in brain samples to assess the age of different types of brain and/or immune cells, including but not limited to microglia, macrophages, glia, and neurons. The disclosure also relates to a method for the measurement of AF in brain samples to assess the type of brain and/or immune cell, including but not limited to microglia, macrophages, glia, and neurons.

[0063] Cell preparation: Cell isolation methods include dissociation/homogenization and digestion of brain tissues of different kind (whole brain, hemibrain, single brain regions) to obtain a single cell suspension. This is achieved using mechanical force, enzyme-based digestion (e.g. papain, trypsin, collagenase) or commercially available kits followed by crude debris removal through a cell strainer (e.g. 40um). Next, Percoll gradients are used to remove myelin debris and enrich microglia. For example, cell pellets from digested brain samples are resuspended in 9ml of 30% Percoll solution in RPMI medium (supplemented with 5% FBS), then gently laid over 1ml of 70% Percoll in RPMI medium (supplemented with 5% FBS) in a 15ml canonical tube. Finally, 1ml of DPBS is added to the top of 30% Percoll solution. Samples are then centrifugated at 800g for 20’ at a recommended temperature of 10C, with maximum acceleration and no brake. After centrifugation, myelin at the DPBS/ 30% Percoll interphase is removed and discarded. Enriched microglia and other types cells at the 70%/30% Percoll interphase are carefully transferred into a new tube for staining.

[0064] Samples: AF differences in brain cells were compared between 3, 9, 15, and 24+ month old (mo.) male and female mice, n = 5 - 6 per age group per sex. [0065] Sample staining: After being washed in RPM1 medium supplemented with 5% FBS, cells are resuspended in FACS buffer with a blocking reagent and incubated on ice for 15 minutes. The next step is the staining with fluorophore-conjugated antibodies to label different types of brain and immune cells, washed, resuspended and loaded to sample tubes. Sample staining was performed differently depending on which instrument was used to acquire AF data. For the ID7000, CD45-APC and CD1 lb-BV421 are used at the concentration of 1 : 100 in FACS buffer for 30 minutes on ice. Samples are then washed and resuspended in FACS buffer. Samples run on the BD FACSymphony A3 were stained with one antibody (CD 1 Ib-APC-eFluor™ 780), followed by the same wash steps as those used on the ID7000.

[0066] Instruments: Two different instruments were used to simultaneously measure AF of cells isolated from both male and female mouse brains of different ages. Some samples were run on an ID7000 spectral analyzer (Sony Biotechnology) equipped with six excitation lasers (320nm, 355nm, 405nm, 488nm, 561nm and 637nm), and 184 detectors. Some samples were run on a FACS analyzer (BD FACSymphony A3) equipped with 5 excitation lasers (355nm, 405nm, 488nm, 561nm and 635nm) and 24 detection channels (U378/29, U515/30, U586/15, U670/30, U740/35, U820/60, UV431/28, UV470/14, UV610/20, UV670/30, UV710/50, UV780/60, B515/20, B610/20, B670/30, B710/50, B780/60, Y586/15, Y610/20, Y670/30, Y780/60, R670/30, R710/50, R780/60).

[0067] Data analysis for ID7000 spectral analyzer: Signature fluorescence spectra were created using instrument analysis software and autofluorescence specifically was located using an internal software application. Specifically, emission spectra were collected from each of the excitation lasers. Five different AF colors were identified across the emission spectrum. “AF colors” are AF signals deemed unique for a particular gated population based upon signal derived from all lasers across all detector channels. Signals from detected AF color channels are recorded and combined to create a full spectral emission signal. Single fluorophore- stained samples (or calibration beads) were used to establish the full emission signature of each fluorophore. These signatures were then used in a process called Spectral Unmixing to calculate and subtract fluorescence spill overs in samples. This spectral signature fully captures cells’ AF spectrum. Spectral unmixing and autofluorescence identification was performed by the Sony ID7000 software, exported as FCS files, and further analyzed using Flowjo software. After gating, fluorescence intensities were measured and plotted using Prism Graphpad software. See Figures 1, 2A, 4, and 5 A. Figures 2B and 5B demonstrate the gating strategies used for the data of Figures 2A and 5A. [0068] Data analysis for BD FACSymphony A3: Brain samples for the BD FACS Symphony A3 were stained with only one antibody, CD1 lb-APC-eF780. This is to avoid the complication from inaccurate compensation of multiple fluorochromes, which leads to interference with AF signals. In the analysis of this set of data, CDllb+ population is considered as microglia, with the caveat of up to 5% of this population being contaminated by macrophages and other immune cells. However, based on the data from TD7000s, AF signal strengths from macrophage and other immune cells are significantly lower than microglia, therefore their AF signals have little contribution to those from microglia (>95% of CD1 lb+ cells). That is, AF signals from CD1 lb+ population from this protocol faithfully reflect AF signals from microglia with minimum interference from the up to 5% contamination. The signals from all detectable channels were collected (data exported as FCS files). Fluorescence intensities were analyzed in FlowJo and plotted using Prism Graphpad software to assess AF signals from microglia. Data are plotted in spectrum view to compare differences between age groups. Normalized AUC (area under curve) is used for quantitative comparison and statistical analyses. See Figures 3 and 6.

[0069] Results from ID7000: An age-dependent increase of AF signal is primarily detected in AF color 3 (and partially AF colors 2 and 6) for both male and female primary mouse microglial cells (Figures 1 and 4).

[0070] A similar age-dependent increase in AF color 3 was detected in brain macrophages from both male and female mice. This is expected as microglia and brain macrophages are both phagocytes of the myeloid lineage. Other brain immune cells and neurons/glia cell populations display a different modulation of AF, that decreases with age in AF color 4 in both male and female mice (Figures 2 A and 5A).

EXAMPLE 2

[0071] Background: As evidence of replicability, a similar analysis was run on a different ID7000 spectral analyzer having seven excitation lasers and 186 detectors.

[0072] Cell isolation: The general cell isolation, dissociation/homogenization, digestion, enrichment, and staining preparation protocols are as described in Example 1.

[0073] Samples: AF differences in brain cells were compared between 3 month and 21 month old (mo.) mice.

[0074] Sample Staining: After being washed in RPMI medium supplemented with 5% FBS, enriched cells are resuspended in FACS buffer with a blocking reagent and incubated on ice for 15 minutes. Microglia are stained using fluorophore-conjugated antibodies. CD45-APC and CD1 lb-BV421 are used at the concentration of 1: 100 in FACS buffer for 30 minutes on ice. Samples are then washed, resuspended in FACS buffer and transferred in 5ml tubes to be analyzed at the spectral analyzer.

[0075] Instrumentation: Samples are then run on an ID7000 spectral analyzer (Sony Biotechnology) equipped with a series of seven excitation lasers (e.g. 320nm, 350nm, 405nm, 488nm, 561 nm, 637nm and 808nm) and 186 detectors.

[0076] Data analysis: Signature fluorescence spectra were created using instrument analysis software and autofluorescence specifically was located using an internal software application. Specifically, microglia AF is measured in multiple channels, including AF channel-4 and AF channel-6. (Note that the numbering of autofluorescing colors/channels differs from Example 1 due to the differences in excitation lasers and accompanying detection channels). Signals from detected channels are recorded and combined to create a full spectral emission signal. Single fluorophore-stained samples (or calibration beads) were used to establish the full emission fingerprint of each fluorophore. These fingerprints were then used in a process called Spectral Unmixing to calculate and subtract fluorescence spill overs in samples. This spectral signature fully captures cells’ AF spectrum and are useful as a proxy of microglia age. Cell gating was performed as shown in Figures 2B and 5B.

[0077] Results: AF is measured in multiple channels — results are shown for autofluorescing channels 1, 2, 4, and 6 (“AF colors” in Figures 7-10). Of note, in comparing Example 2 to Example 1, different AF colors were observed as being modulated by aging in the four brain cell populations. This is expected as the two instruments that were used have different numbers of lasers and detectors, features that influence the ability of identifying AF colors. In addition, cell type specific AF channels were identified for macrophages, neurons and glia, and other immune cells.

[0078] Microglia from old mice display increased intensity of AF colors 4 and 6 (Figure 7A). This effect is similar in both male (Figure 7B) and female (Figure 7C) old mice when compared to the sex-matched young counterpart. Differently, age increased AF colors 2 and 4 in brain macrophages (Figure 8A). Similar trends were observed in both male (Figure 8B) and female (Figure 8C) mice. Other immune cells showed an age-dependent increase in AF specific for AF color 4 (Figure 9A). No sex differences were observed (Figures 9B and 9C). AF color 6 increased moderately in the neuron and glia populations of old mice (Figure 10A). Interestingly, AF color 1 decreased in the neuron and glia population of old female mice (Figure 10C) and show a similar trend in the males (Figure 10B). EXAMPLE 3

[0079] Microglia specific autofluorescence (AF) in young (5mo) vs. aged (20m) brain slices using AiryScan 980 Spectral Scanner (Zeiss) is shown in Figure 11. Microglia cells are stained with AF647 conjugated Ibal antibody and detected by PMTs that collect emission signals that peak at 685nm and 805nm under the excitation of the 639nm laser. The full emission spectrum of microglia (Ibal positive cells) excited by the 488nm laser is collected and compared between young and aged brains. Background emission spectra from non-microglia cells (Ibal negative cells) are also collected and plotted to show the specificity of Ibal staining. Microglia from aged brains in both the corpus callosum (left panel) and the hippocampus (right panel) show significant increased autofluorescence compared to those from young brains. These results demonstrate that spectral imaging based measurement can be used to detect age-dependent difference of autofluorescence in a cell type specific manner.

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7. Croce AC, Bottiroli G. Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. Eur J Histochem. 2014 Dec 12;58(4):2461. doi: 10.4081/ejh.2014.2461. PMID: 25578980; PMCID: PMC4289852.

EXEMPLARY ASPECTS

[0050] Implementation 1 : A method of probing a cellular characteristic, the method comprising preparing a biological specimen for a spectral analysis, the biological specimen comprising at least one analytical target, exciting the biological specimen with at least one excitation laser, the excitation laser having an excitation wavelength, creating an emission spectrum related to the excitation wavelength by detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum, collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum, establishing an AF spectrum related to the analytical target using the autofluorescence measurements from across the emission spectrum, and using the AF spectrum to probe a cell age.

[0051] Implementation 2: A method of probing a cellular characteristic, the method comprising preparing a biological specimen for a spectral analysis, the biological specimen comprising at least one analytical target, exciting the biological specimen with at least one excitation laser, the excitation laser having an excitation wavelength, creating an emission spectrum related to the excitation wavelength by detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum, collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum, establishing an AF spectrum related to the analytical target using the autofluorescence measurements from across the emission spectrum, and using the AF spectrum to probe a cell type.

[0052] Implementation 3 : The method according to any implementation herein, particularly either implementation 1 or implementation 2, wherein the at least one excitation laser comprises a plurality of excitation lasers, each excitation laser of the plurality of excitation lasers having a differing excitation wavelength than the other excitation lasers of the plurality of excitation lasers.

[0053] Implementation 4: The method according to any implementation herein, particularly implementations 1-3, further comprising creating a plurality of emission spectrums, each emission spectrum of the plurality of emission spectrums related to a particular excitation wavelength from the plurality of excitation lasers.

[0054] Implementation 5 : The method according to any implementation herein, particularly implementations 1-4, wherein the plurality of excitation lasers comprises six or more excitation lasers.

[0055] Implementation 6: The method according to any implementation herein, particularly implementations 1-5, the method further comprising sequentially stimulating the biological specimen by exciting the biological specimen with a first excitation laser of the plurality of excitation lasers, pausing from 15 to 65 microseconds, then exciting the biological specimen with a next excitation laser of the plurality of excitation lasers, thereby creating a sequential stimulation.

[0056] Implementation 7 : The method according to any implementation herein, particularly implementation 6, wherein the total duration of the sequential stimulation is from 25 to 600 microseconds.

[0057] Implementation 8: The method according to any implementation herein, particularly implementations 1-7, wherein detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum comprises detecting emission in at least two of the ultraviolet, visible, and infrared ranges.

[0058] Implementation 9: The method according to any implementation herein, particularly implementations 1-8, wherein the biological specimen comprises a cell suspension.

[0059] Implementation 10: The method according to any implementation herein, particularly implementations 1-8, wherein the biological specimen comprises a tissue sample.

[0060] Implementation 11: The method according to any implementation herein, particularly implementations 1-8, wherein the biological specimen comprises a bodily fluid.

[0061] Implementation 12: The method according to any implementation herein, particularly implementations 1-11, wherein preparing the biological specimen comprises preparing a slide. [0062] Implementation 13: The method according to any implementation herein, particularly implementations 1-12, wherein preparing the biological specimen comprises preparing a cell suspension for a spectral cytometry assay. [0063] Implementation 14: The method according to any implementation herein, particularly implementations 1-13, wherein the analytical target is a cell population.

[0064] Implementation 15: The method according to any implementation herein, particularly implementation 14, wherein the cell population is a mammalian cell population.

[0065] Implementation 16: The method according to any implementation herein, particularly implementations 1 -15, wherein the analytical target is a cellular component.

[0066] Implementation 17: The method according to any implementation herein, particularly implementation 16, wherein the cellular component comprises a membrane, an organelle, or an extracellular matrix material.

[0067] Implementation 18: The method according to any implementation herein, particularly implementations 1-17, wherein collecting AF measurements from the analytical target comprises identifying emission signals from the analytical target.

[0068] Implementation 19: The method according to any implementation herein, particularly implementations 1-18, wherein establishing an AF spectrum related to the analytical target comprises applying a spectral unmixing algorithm.

[0069] Implementation 20: The method according to any implementation herein, particularly implementations 1-19, wherein using the AF spectrum to probe a cellular characteristic comprises comparing the AF spectrum related to the analytical target to at least one control AF value.

[0070] Implementation 21: The method according to any implementation herein, particularly implementations 1-20, wherein the at least one control AF value is from a different biological specimen with known properties.

[0071] Implementation 22: The method according to any implementation herein, particularly implementations 1-21, wherein the control AF value is predetermined.

[0072] Implementation 23: The method according to any implementation herein, particularly implementations 1-22, wherein the cellular characteristic is a cell health.

[0073] Implementation 24: The method according to any implementation herein, particularly implementations 1-23, wherein the cellular characteristic is a response to a treatment.

[0074] Implementation 25: The method according to any implementation herein, particularly implementations 1-24, wherein the method is performed without applying a cellular stain to the biological specimen.

[0075] Implementation 26: The method according to any implementation herein, particularly implementations 1-25, wherein the spectral analysis utilizes spectral cytometry. [0076] Implementation 27 : The method according to any implementation herein, particularly implementations 1-26, wherein the spectral analysis utilizes fluorescence microscopy.

[0077] The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The implementation was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various implementations with various modifications as are suited to the particular use contemplated.

Claims

WHAT IS CLAIMED IS:

1. A method of probing a cellular characteristic, the method comprising: preparing a biological specimen for a spectral analysis, the biological specimen comprising at least one analytical target; exciting the biological specimen with at least one excitation laser, the excitation laser having an excitation wavelength; creating an emission spectrum related to the excitation wavelength by detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum; collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum; establishing an AF spectrum related to the analytical target using the autofluorescence measurements from across the emission spectrum; and using the AF spectrum to probe at least one cellular characteristic, wherein the at least one cellular characteristic comprises a cell age.

2. A method of probing a cellular characteristic, the method comprising: preparing a biological specimen for a spectral analysis, the biological specimen comprising at least one analytical target; exciting the biological specimen with at least one excitation laser, the excitation laser having an excitation wavelength; creating an emission spectrum related to the excitation wavelength by detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum; collecting autofluorescence (AF) measurements from the analytical target across the emission spectrum; establishing an AF spectrum related to the analytical target using the autofluorescence measurements from across the emission spectrum; and using the AF spectrum to probe at least one cellular characteristic, wherein the at least one cellular characteristic comprises a cell type.

3. The method of either claim 1 or claim 2, wherein the at least one excitation laser comprises a plurality of excitation lasers, each excitation laser of the plurality of excitation lasers having a differing excitation wavelength than the other excitation lasers of the plurality of excitation lasers.

4. The method of any one of claims 1-3, further comprising creating a plurality of emission spectrums, each emission spectrum of the plurality of emission spectrums related to a particular excitation wavelength from the plurality of excitation lasers.

5. The method of any one of claims 1-4, wherein the plurality of excitation lasers comprises six or more excitation lasers.

6. The method of any one of claims 1-5, further comprising sequentially stimulating the biological specimen by exciting the biological specimen with a first excitation laser of the plurality of excitation lasers, pausing from 15 to 65 microseconds, then exciting the biological specimen with a next excitation laser of the plurality of excitation lasers, thereby creating a sequential stimulation.

7. The method of claim 6, wherein the total duration of the sequential stimulation is from 25 to 600 microseconds.

8. The method of any one of claims 1-7, wherein detecting emission signals from the biological specimen at a plurality of wavelengths across the electromagnetic (EM) spectrum comprises detecting emission in at least two of the ultraviolet, visible, and infrared ranges.

9. The method of any one of claims 1-8, wherein the biological specimen comprises a cell suspension.

10. The method of any one of claims 1-8, wherein the biological specimen comprises a tissue sample.

11. The method of any one of claims 1-8, wherein the biological specimen comprises a bodily fluid.

12. The method of any one of claims 1-11, wherein preparing the biological specimen comprises preparing a slide.

13. The method of any one of claims 1-12, wherein preparing the biological specimen comprises preparing a cell suspension for a spectral cytometry assay.

14. The method of any one of claims 1-13, wherein the analytical target is a cell population.

15. The method of claim 14, wherein the cell population is a mammalian cell population.

16. The method of any one of claims 1-15, wherein the analytical target is a cellular component.

17. The method of claim 16, wherein the cellular component comprises a membrane, an organelle, or an extracellular matrix material.

18. The method of any one of claims 1-17, wherein collecting AF measurements from the analytical target comprises identifying emission signals from the analytical target.

19. The method of any one of claims 1-18, wherein establishing an AF spectrum related to the analytical target comprises applying a spectral unmixing algorithm.

20. The method of any one of claims 1-19, wherein using the AF spectrum to probe a cellular characteristic comprises comparing the AF spectrum related to the analytical target to at least one control AF value.

21. The method of any one of claims 1-20, wherein the at least one control AF value is from a different biological specimen with known properties.

22. The method of any one of claims 1-21, wherein the control AF value is predetermined.

23. The method of any one of claims 1-22, wherein the at least one cellular characteristic further comprises a cell health.

24. The method of any one of claims 1-23, wherein the at least one cellular characteristic further comprises a response to a treatment.

25. The method of any one of claims 1-24, wherein the method is performed without applying a cellular stain to the biological specimen.

26. The method of any one of claims 1-25, wherein the spectral analysis utilizes spectral cytometry.

27. The method of any one of claims 1-26, wherein the spectral analysis utilizes spectral fluorescence microscopy.