# US 11,906,432 B2 · Enhanced, rapid, homogeneous, cell staining and assay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,906,432 B2 - Document ID: US11906432B2 - Status: Granted - Kind: B2 - Application number: 17/268,799 - Filed: 2019-08-23 - Published: 2021-08-12 - Granted: 2024-02-20 - Earliest claimed: 2018-08-23 - Assignees: Essenlix Corporation - Applicants: Essenlix Corporation - Inventors: Stephen Y. Chou; Wei Ding; Ji Li; Shengjian Cai; Yufan Zhang; Jia Peng - Web page: https://patentgrep.com/patent/US11906432B2 ## CPC - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} - G01N 21/76: Chemiluminescence; Bioluminescence - G01N 33/54373: {involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings} - G01N 2333/195: from bacteria - G01N 2333/70514: CD4 - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention provides, among other things, devices, kits, apparatus, and methods for rapid homogenous cell staining and imaging. Particularly, in some embodiments, the present invention can immunochemically stain a cell or a tissue in less than 60 seconds without washing. In some embodiments, the present invention stains and observers analyte (protein or nucleic acid) inside a cell in 60 seconds without wash. ## Claims ### Claim 1 A method of enhancing a homogenous detection of an analyte in a cell that is in a sample, comprising: (a) providing a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample comprising a cell that contains or is suspected of containing an analyte, (b) providing a detection probe that specifically binds the analyte and is capable of emitting a light at a wavelength, wherein the detection probe diffuses in the sample; (c) providing an optical enhancer that diffuses in the sample, binds to the cell, and is capable of emitting a light of a wavelength that overlaps with or within a 150 nm from the wavelength of the light emitted by the detection probe; (d) sandwiching the sample, the detection probe, and the optical enhancer between the two sample contact areas of the two plates to form a thin layer of a thickness of 200 microns (um) or less; and (e) imaging, using an imager, after step (d) and without using any washing step, the thin layer to detect the cell that has the analyte bound to the detection probe; wherein the thin layer has a thickness that is selected so that for a given concentration of the cell in the thin layer, each individual cell does not substantially overlap other cells in the imaging; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is selected to make, in step (e) of imaging, in the thin layer, the location having a bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the cell. ### Claim 2 (depends on 1) The method of claim 1, wherein the detection probe is deposited on at least one of the sample contact areas. ### Claim 3 (depends on 1) The method of claim 1, wherein the detection probe and the optical enhancer are deposited on at least one of the sample contact areas. ### Claim 4 (depends on 1) The method of claim 1, wherein the imaging further comprises a step of machine learning in detecting the analyte. ### Claim 5 (depends on 1) The method of claim 1, wherein the optical enhancer is a cell permeant dye that readily penetrates a cell wall and stains the components of the same without requiring the additional presence of a permeabilizing agent. ### Claim 6 (depends on 1) The method of claim 1, wherein the analyte comprises a molecule, cells, tissues, viruses, or nanoparticles with different shapes, wherein the molecule comprising a protein, peptides, DNA, RNA, or nucleic acid. ### Claim 7 (depends on 1) The method of claim 1, wherein the cell is a prokaryote or eukaryote. ### Claim 8 (depends on 1) The method of claim 1, wherein the cell in a sample has a quantity of one or more than one. ### Claim 9 (depends on 1) The method of claim 1, wherein the detection probe is a label, wherein the label is immunoassay antibody label, RNA label, or staining dye and is coated on the plate with a final concentration in sample of 0.05 mg/mL, 0.15 mg/mL, 0.3 mg/mL, 0.5 mg/mL, 1.0 mg/mL, or in a range between any of the two values. ### Claim 10 (depends on 1) The method of claim 1, wherein the detection probe is a fluorescent RNA probe. ### Claim 11 (depends on 1) The method of claim 1, wherein an average thickness of the layer is about equal to a minimum dimension of the analyte in the sample. ### Claim 12 A device for enhancing a homogenous detection of an analyte in a cell that is in a sample, comprising: (a) a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample comprising a cell that contains or is suspected of containing an analyte, (b) a detection probe that specifically binds the analyte and is capable of emitting a light at a wavelength, wherein the detection probe diffuses in the sample; (c) an optical enhancer that binds to the cell and emits a light of a wavelength that overlaps with or within a 150 nm from the wavelength of the light emitted by the detection probe; wherein the first and second plates sandwich the sample, the detection probe, and the optical enhancer between the two sample contact areas to form a thin layer of a thickness of 200 microns (um) or less; and wherein the thin layer has a thickness is selected so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells for an imaging; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is selected to make, in the imaging, in the thin layer, the location having a bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the cell. ### Claim 13 (depends on 12) The device of claim 12, further comprising an imager that images the thin layer to detect the bound detection probe. ### Claim 14 (depends on 12) The device of claim 12, wherein the optical enhancer is coated on one or both of the sample contact areas. ### Claim 15 (depends on 12) The device of claim 12, wherein the thin layer has a thickness selected to make, in the step (e) of imaging, in the thin layer, the location having the bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the cell, the analyte is a nucleic acid and the detection probe is a probe for fluorescence in-situ hybridization (FISH). ### Claim 16 (depends on 12) The device of claim 12, wherein the detection probe is an immune probe. ### Claim 17 (depends on 12) The device of claim 12, wherein the analyte is a CD4. ### Claim 18 (depends on 12) The device of claim 12, wherein the first plate and the second plate are movable relative to each other into a different configuration, including an open configuration and a closed configuration, wherein: in the open configuration, the first plate and the second plate are partially or entirely separated, and the sample is deposited in the sample contact area on one or both of the plates, and a separation sheet is removed from any contact with one or both of the plates; and in the closed configuration at least part of the deposited sample is compressed by the two plates into a thin layer. ### Claim 19 (depends on 18) The method of claim 18, wherein at the closed configuration, the thickness of the thin layer is thin enough to enable the device to analyze the sample in 60 seconds or less. ### Claim 20 (depends on 12) The device of claim 12, further comprising spacers, wherein: (i) the first plate and the second plate are movable relative to each other into a different configuration, including an open configuration and a closed configuration, and (ii) one or both of the plates comprise spacers that are fixed with a respective plate, wherein in an open configuration, the two plates are either completely or partially separated apart, the spacing between the plates is not regulated by the spacers, and the sample is deposited on one or both of the plates; and wherein in the closed configuration, which is configured after the sample deposition in the open configuration, at least a part of the sample is between the two plates and in contact with the two plates, and has a highly uniform thickness that is regulated by the spacers and the two sample surfaces of the plates and is equal to or less than 150 um. ### Claim 21 (depends on 20) The device of claim 20, wherein the spacers have a height of 30 um or less. ### Claim 22 (depends on 20) The device of claim 20, wherein the spacers have a height of 10 um or less. ### Claim 23 (depends on 20) The device of claim 20, wherein the spacers have a height of 2 um or less. ### Claim 24 (depends on 20) The device of claim 20, wherein the spacers have an inter-spacer distance in the range of 1 um to 200 um. ### Claim 25 (depends on 20) The device of claim 20, wherein the spacers have an inter-spacer distance in the range of 200 um to 1000 um. ### Claim 26 (depends on 20) The device of claim 20, wherein the detection probe is a fluorescent RNA probe. ### Claim 27 (depends on 26) The device of claim 26, wherein the spacers have a inter-space distance (ISD), a fourth power of the ISD divided by the thickness of the flexible plate (h) and the Young's modulus (E) of the flexible plate, ISD4/(hE), is equal to or less than 106 um3/GPa; and the thickness of the flexible plate times the Young's modulus of the flexible plate is in the range 60 to 750 GPa-um. ### Claim 28 (depends on 20) The device of claim 20, wherein the spacers function as a location marker, a scale marker, an imaging marker, or any combination thereof. ### Claim 29 (depends on 20) The device of claim 20, wherein the spacers have an inter-spacer distance in the range of 1 um to 50 um. ### Claim 30 (depends on 20) The device of claim 20, wherein the spacers have an inter-spacer distance in the range of 50 um to 120 um. ### Claim 31 (depends on 20) The device of claim 20, wherein the spacers have an inter-spacer distance in the range of 120 um to 200 um. ### Claim 32 (depends on 20) The device of claim 20, wherein the spacers have an inter-spacer distance that is substantially periodic. ### Claim 33 (depends on 20) The device of claim 20, wherein the spacers are pillars with a cross-sectional shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, and any combination of the same. ### Claim 34 (depends on 20) The device of claim 20, wherein the spacers have a pillar shape and have a substantially flat top surface, wherein, for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1. ### Claim 35 (depends on 20) The device of claim 20, wherein a minimum lateral dimension of the spacer is in the range of 0.5 um to 100 um. ### Claim 36 (depends on 12) The device of claim 12, further comprising a non-volatile storage medium that has an algorithm of machine learning for detecting the analyte. ### Claim 37 (depends on 12) The device of claim 12, wherein the thickness of the thin layer is 2 um or less. ### Claim 38 (depends on 12) The device of claim 12, wherein the sample is from a biological sample, an environmental sample, a chemical sample, or a clinical sample. ### Claim 39 (depends on 12) The device of claim 12, further comprising a reagent coated on the device for making protein cross link, the reagent comprises formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate, or potassium permanganate. ### Claim 40 (depends on 12) The device of claim 12, wherein the optical enhancer emits a light of a wavelength that overlaps with or within a 30 nm from the wavelength of the light emitted by the detection probe. ### Claim 41 (depends on 12) The device of claim 12, wherein the device further comprises, on one or both plates, multi reagent layers including anti-conglutination, cell lysing, cell staining, release time control material layers, and their combinations. ### Claim 42 (depends on 41) The device of claim 41, wherein the anti-conglutination agent comprises ethylenediaminetetraacetic acid (EDTA), EDTA disodium, dipotassium ethylenediaminetetraacetic acid, or tripotassium ethylenediaminetetraacetic acid. ### Claim 43 (depends on 12) The device of claim 12, wherein one or both sample areas are coated with the cell stain agent comprising Wright's stain, Giemsa stain, May-Grünwald stain, Leishman's stain, Erythrosine B stain, and other fluorescence stain including Acridine orange dye, 3,3-dihexyloxacarbocyanine, Propidium Iodide, Fluorescein Isothiocyanate and Basic Orange 21) dye, Ethidium Bromide, Brilliant Sulfaflavine and a Stilbene Disulfonic Acid derivative, Erythrosine B or trypan blue, Hoechst 33342, Trihydrochloride, Trihydrate, and 4′,6-Diamidino-2-Phenylindole, and YOYO. ### Claim 44 (depends on 12) The device of claim 12, further comprising a release time control material coated on the first or second plate, the release time control material comprises albumin, carbomers, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinylpyrrolidone, or polyvinyl alcohol. ### Claim 45 (depends on 12) The device of claim 12, wherein one or both sample areas are coated with the chemicals comprising Surfactant, Zwittergent, 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate (ASB-14), 3-[N,N-Dimethyl(3-palmitoylaminopropyl) ammonio]-propanesulfonate (ASB-16), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Cationic surfactant NN-[Tris(hydroxymethyl) methyl]-N-alkyl-N,N-dimethyl ammonium chloride (IIa), IIb, IIc, IId, cetyltrimethylammonium chloride (CTAC), polyoxyethylene sorbitan monolaurate (TWEEN® 20), polyoxyethylene sorbitan monopalmitate (TWEEN® 40), polyoxyethylene sorbitan monostearate (TWEEN® 60), polyoxyethylene sorbitan monooleate (TWEEN® 80), Sodium lauryl sulfate (SLS), ammonium lauryl sulfate, cetyltrimethylammonium bromide (CTAB), sodium lauryl ether sulfate (SLES), sodium myreth sulfate, docusate, perfluorooctanesulfonate, alkyl-aryl ether phosphates, alkyl ether phosphates, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, dioctadecyldimethlyammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow-range ethoxylate, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (TRITON X-100™), polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamers, glycerol monostearate, glycerol monolaurate, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, decyl glucoside, lauryl glucoside, octyl glucoside, lauryldimethylamine oxide, dimethyl sulfoxide, or phosphine oxide. ### Claim 46 (depends on 12) The device of claim 12, wherein one or both sample areas are coated with a reagent comprising non-ionic copolymer surfactant (Pluronic F-127), polyoxyl 35 castor oil (Cremophor EL), Polyoxyethylene-polyoxypropylene block copolymer (Pluronic F-68), Polyoxyethylene (40) stearate (Myrj 52), Polyoxyethylene lauryl ether (Brij 35), sodium oleate, sodium dodecyl sulfate, TWEEN® 20, TWEEN® 40, TWEEN® 60, TWEEN® 80, SLS, CTAB, CTAC, Tamoxifen, saponin, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, quinine compounds, arsenic, dapsone, chromium/chromates, platinum salts, nickel compounds, copper, lead, cis-platinum, nitrites, nitrofurantoin, penicillin, phenazopyridine rho immune globulin, ribavirin, sulfonamides, sulfones. ### Claim 47 (depends on 12) The device of claim 12, wherein the detection probe is a label, wherein the label is immunoassay antibody label, RNA label, or staining dye and is coated on the plate with a final concentration in sample of 0.1 nM/mL, 0.5 nM/mL, 1 nM/mL, 5 nM/mL, 10 nM/mL, 15 nM/mL, 20 nM/mL, 50 nM/mL, or in a range between any of the two values. ### Claim 48 (depends on 12) The device of claim 12, wherein the thickness of the sample is 30 um or less. ### Claim 49 (depends on 12) The device of claim 12, wherein the thickness of the sample is 10 um or less. ### Claim 50 (depends on 12) The device of claim 12, wherein the concentration of the detection probe in the sample is 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 uM, 10 nM, 50 nM, 100 nM or in a range between any of the two values. ### Claim 51 (depends on 12) The device of claim 12, wherein the concentration of probe in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 M, or in a range between any of the two values. ### Claim 52 (depends on 12) The device of claim 12, wherein the concentration of the optical enhancer in the sample is 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 500 μM, or in a range between any of the two values. ### Claim 53 (depends on 12) The device of claim 12, wherein the concentration of the optical enhancer in the sample is 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, or in a range between any of the two values. ### Claim 54 (depends on 12) The device of claim 12, wherein the optical enhancer's light wavelength and the probe's light wavelength is within 1 nm, 5 nm, 10 nm, 20 nm, 50 nm, or 100 nm. ### Claim 55 (depends on 12) The device of claim 12, wherein the optical enhancer's light wavelength and the probe's light wavelength is within 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, or 50 nm. ### Claim 56 (depends on 12) The device of claim 12, wherein one or both plates comprises a scale marker, either on a surface of or inside the plate, that provides information of a lateral dimension of a structure of the sample and/or the plate. ### Claim 57 (depends on 12) The device of claim 12, wherein one or both of the sample contact surfaces comprises a scale marker, a location marker, or an imaging marker. ### Claim 58 (depends on 12) The device of claim 12, wherein the first plate and the second plate are connected by a hinge. ### Claim 59 (depends on 12) The device of claim 12, wherein the thickness of the thin layer is so thin that the saturation incubation time of the sample with the detection probe is 60 seconds or less. ### Claim 60 (depends on 12) The device of claim 12, wherein the binding of the optical enhancer to the cell is specific. ### Claim 61 (depends on 12) The device of claim 12, wherein the binding of the optical enhancer to the cell is nonspecific. ### Claim 62 (depends on 12) The device of claim 12, wherein the binding of the optical enhancer to the target cell is nonspecific, wherein the optical enhancer binds to nucleic acids of the target cell. ### Claim 63 (depends on 12) A system of enhancing a homogenous detection of an analyte in a cell that is in a sample, comprising: (a) the device of claim 12; and (b) a communication device, wherein the communication device comprising (i) one or a plurality of cameras for the detecting signal and/or imaging the sample; and (ii) electronics, signal processors, hardware and software for receiving and/or processing the detected signal and/or the image of the sample. ### Claim 64 (depends on 63) The device of claim 63, wherein the communication device is further configured to communicate information of the test and the subject to a cloud network, the cloud network is configured to process the information to refine the test results, and the refined test results will send back the subject. ### Claim 65 (depends on 63) The device of claim 63, wherein the communication device is configured to receive a prescription, diagnosis or a recommendation from a medical professional. ### Claim 66 A device for a rapid homogenous detection of an analyte inside a membrane of a cell in a sample, comprising: (a) a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample comprising a cell that contains or is suspected of containing an analyte inside of the cell membrane; (b) a detection probe that specifically binds the analyte and is capable of emitting a light at a wavelength, wherein the detection probe diffuses in the sample; (c) a permeabilization agent that makes a membrane of the cell permeable to the detection probe; wherein the first and second plates are configured to sandwich the sample, the detection probe, and the permeabilization agent between the two sample contact areas of the two plates to form a thin layer of a thickness of 200 microns (um) or less; wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in an imaging, wherein the imaging is performed without a washing step; wherein the thickness of the thin layer and the concentration of the detection probe in the sample, are configured to make, in the imaging, in the thin layer, the location having the detection probe that bound to the analyte inside the cell membrane is distinguishable from the locations that do not have the cell; and wherein the washing step is a step for removing unbound detection probe, permeabilization reagent, or both from the sample contact areas. ### Claim 67 (depends on 66) The device of claim 66, further comprising an optical enhancer, wherein the first and second plates are configured to sandwich the sample, the detection probe, the permeabilization agent, and the optical enhancer between the two sample contact areas of the two plates to form a thin layer of a thickness of 200 microns (um) or less; wherein the optical enhancer diffuses in the sample, binds to the cell, and is capable of emitting a light of a wavelength that overlaps with or within a 150 nm from the wavelength of the light emitted by the detection probe; wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in an imaging, wherein the imaging is performed without a washing step; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured to make, in the step (e) of imaging, in the thin layer, the location having the bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the cell; and wherein the washing step is a step of removing unbound detection probe, permeabilization reagent, or both from the sample contact areas. ### Claim 68 (depends on 67) The device of claim 67, wherein the detection probe, the permeabilization agent, or the optical enhancer is deposited on one or both of the sample surfaces. ### Claim 69 (depends on 66) The device of claim 66, wherein the permeabilization agent makes cell membrane or cell nucleus permeable, comprising TRITON X-100™, surfactant, Zwittergent, 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate (ASB-14), 3-[N,N-Dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate (ASB-16), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Cationic surfactant NN-[Tris(hydroxymethyl) methyl]-N-alkyl-N,N-dimethyl ammonium chloride (IIa), IIb, IIc, IId, CTAC, TWEEN® 20, TWEEN® 40, TWEEN® 60, TWEEN® 80, Sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), sodium myreth sulfate, docusate, perfluorooctanesulfonate, alkyl-aryl ether phosphates, alkyl ether phosphates, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, dioctadecyldimethlyammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow-range ethoxylate, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, TRITON X-100™, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamers, glycerol monostearate, glycerol monolaurate, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, decyl glucoside, lauryl glucoside, octyl glucoside, lauryldimethylamine oxide, dimethyl sulfoxide, phosphine oxide. ### Claim 70 (depends on 69) The device of claim 69, wherein the Zwittergent is coated onto the first plate, or the second plate or both. ### Claim 71 (depends on 70) The device of claim 70, wherein one or both sample areas are coated with Zwittergent of an area concentration of 3 ng/mm2, 5 ng/mm2, 8 ng/mm2, 12 ng/mm2, 15 ng/mm2, 25 ng/mm2, 35 ng/mm2, 50 ng/mm2, 80 ng/mm2, 100 ng/mm2 or in a range between any of the two values. ### Claim 72 (depends on 66) The device of claim 66, wherein permeabilization agent is an agent making the cell membrane or cell nucleus of the cell permeable, by causing osmotic pressure difference between the cell membrane as dilution, or tuning salt concentration. ### Claim 73 (depends on 66) The device of claim 66, wherein acridine orange is coated onto the first plate, or the second plate or both. ### Claim 74 (depends on 66) The device of claim 66, wherein Methylene blue and Zwittergent is coated onto the first plate, or the second plate or both. ### Claim 75 (depends on 66) The device of claim 66, wherein acridine orange and Zwittergent is coated onto the first plate, or the second plate or both. ### Claim 76 (depends on 75) The device of claim 75, wherein one or both sample areas are coated with the acridine orange of an area concentration of 3 to 10 ng/mm2 and Zwittergent is coated on the plate with an area concentration of 3 to 30 ng/mm2. ### Claim 77 (depends on 66) The device of claim 66, wherein YOYO dye and Zwittergent is coated onto the first plate, or the second plate or both. ### Claim 78 (depends on 66) The device of claim 66, wherein one or both sample areas are coated with the cell stain agent comprising Wright's stain, Giemsa stain, May-Grünwald stain, Leishman's stain, Erythrosine B stain, and other fluorescence stain including Acridine orange dye, 3,3-dihexyloxacarbocyanine (DiOC6), Propidium Iodide (PI), Fluorescein Isothiocyanate (FITC) and Basic Orange 21 (B021) dye, Ethidium Bromide, Brilliant Sulfaflavine and a Stilbene Disulfonic Acid derivative, Erythrosine B or trypan blue, Hoechst 33342, Trihydrochloride, Trihydrate, and DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride), YOYO, acid fuchsine, hematoxylin, Hoechst stains including Hoechst 33258 and Hoechst 33342, methyl green, methylene blue, Nile blue, Nile red, osmium tetraoxide, rhodamine, safranine, MeOSuc-AAPV-AMC, CFSE, BCECF/AM, silver nitrate, neutral red, pyronin Y, Calcein-AM, Dihydroethidium, Xylene Cyanol FF, Rhodamine 123, 4-Methylumbelliferyl palmitate, Fast Blue B Salt, Lucifer Yellow CH dipotassium salt, DAPI dilactate, or Propidium Iodide. ### Claim 79 (depends on 66) The device of claim 66, wherein the detection probe is a label, wherein the label is immunoassay antibody label, RNA label, or staining dye and is coated on the plate with a final concentration in sample of 1 nM/mL, 5 nM/mL, 10 nM/mL, 15 nM/mL, 20 nM/mL, or in a range between any of the two values. ### Claim 80 A method for a rapid homogenous detection of an analyte inside a membrane of a cell in a sample, comprising: (a) providing a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample comprising a cell that contains or is suspected of containing an analyte inside of the cell membrane; (b) providing a detection probe that specifically binds the analyte and is capable of emitting a light at a wavelength, wherein the detection probe diffuses in the sample; (c) providing a permeabilization agent that makes a membrane of the cell permeable to the detection probe; (d) sandwiching the sample, the detection probe, and the permeabilization agent between the two sample contact areas of the two plates to form a thin layer of a thickness of 200 microns (um) or less; and (e) imaging, after the step (d) and without a washing step, the thin layer to detect the cell that has the analyte bound to the detection probe; wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in the imaging; wherein the thickness of the thin layer and the concentration of the detection probe in the sample, are configured to make, in the thin layer, the location having the detection probe that bound to the analyte inside the cell membrane is distinguishable from the locations that do not have the cell; and wherein the washing step is a step for removing unbound detection probe, permeabilization reagent, or both from the sample contact areas. ### Claim 81 (depends on 80) The method of claim 80, further comprising a label, wherein the label is immunoassay antibody label, RNA label, or staining dye and is coated on the plate with a final concentration in sample of 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 5 mg/mL, 10 mg/mL, 50 mg/mL, or in a range between any of the two values. ### Claim 82 A method of a homogenous detection of a Gram positive cell or Gram negative cell in a sample, comprising: (a) providing a first plate and a second plate, each has a sample contact area on its surface, wherein the sample contact surfaces contact a sample that contains or is suspected of containing a Gram positive cell or Gram negative cell; (b) depositing, on at least one of the sample contact areas, a Gram positive stain, a Gram negative stain, or both, wherein the Gram positive and Gram negative stains have two distinguishable colors; wherein the Gram positive stain stains only Gram positive cells while the Gram negative stain stains both Gram positive and negative cells; wherein, in a Gram positive cell that is stained by both Gram positive and negative stains, the Gram positive stain is distinguishable from the Gram negative stain; (c) sandwiching the sample between the two sample contact areas of the two plates to make the sample forming a thin layer of a thickness of 150 microns (um) or less; and (d) imaging using an imager, after the step (c) and without using any washing step, the thin layer to detect cells that are stained by (i) Gram negative stain only, (ii) Gram positive stain; and (iii) both; wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in the imaging; wherein a Gram positive cells displays Gram positive stain color, and a Gram negative cell displays Gram negative stain color only; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured to make, in the step (e) of imaging, in the thin layer, the location having the bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the sample. ### Claim 83 (depends on 82) The method of claim 82, further comprising a label, wherein the label is immunoassay antibody label, RNA label, or staining dye and is coated on the plate with a final concentration in sample of 100 mg/mL, 200 mg/mL, 300 mg/mL, or in a range between any of the two values. ### Claim 84 (depends on 80) The method of claim 80, further comprising an optical enhancer, wherein the first and second plates are configured to sandwich the sample, the detection probe, the permeabilization agent, and the optical enhancer between the two sample contact areas of the two plates to form a thin layer of a thickness of 200 microns (μm) or less; wherein the optical enhancer diffuses in the sample, binds to the cell, and is capable of emitting a light of a wavelength that overlaps with or within a 150 nm from the wavelength of the light emitted by the detection probe; wherein the thin layer sample thickness is configured so that for a given concentration of the cell in the sample, each individual cell does not substantially overlap other cells in an imaging, wherein the imaging is performed without a washing step; wherein the thickness of the thin layer, the concentration of the detection probe in the sample, or the concentration of the optical enhancer in the sample is configured to make, in the step (e) of imaging, in the thin layer, the location having the bound detection probe is distinguishable from the locations not having the bound detection probe, wherein the bound detection probe is the detection probe bound to the analyte in the cell; and wherein the washing step is a step of removing unbound detection probe, permeabilization reagent, or both from the sample contact areas. ### Claim 85 (depends on 84) The method of claim 84, wherein the detection probe, the permeabilization agent, or the optical enhancer is deposited on one or both of the sample surfaces.