Method and system for determining spatial position of biological composition in sample

Abstract

The present application proposes a method for determining the spatial position of one or more biological components (12) in a biological sample (10). The method comprises: optically encoding (S100) biological compositions (12) in a biological sample (10) by a plurality of fluorophores (31, 32, 33, 34) to generate a combination of active fluorophore concentrations indicative of the spatial position of one or more biological compositions (12); separating (S200) the biological composition (12) from the biological sample (10) to produce a separated biological composition (12) of the biological composition (12); and optically decoding (S300) by measuring (S310) the fluorescence of the separated biological composition (12) and mapping (S320) to the spatial position of the biological composition (12) in the unseparated sample (10).

Description

[0001] References to other applications This application claims priority from European patent application EP22383262.7 filed on December 22, 2022. Technical Field

[0002] The present invention relates to a method and system for determining the spatial location of biological components in a biological sample. Background Art

[0003] In the fields of tissue biology and cancer, it is desirable to understand the spatiotemporal distribution of biological components, including cellular machinery, genomic status, metabolites, and gene activity, in order to understand tissue biology and cancer. To this end, a variety of spatial omics technologies have been developed to attempt to measure genomic sequence, protein levels, epigenetic marker levels, metabolite levels, and gene expression levels in different regions of a biological sample.

[0004] Known use has known spatial distribution markers in biological samples.In spatial transcriptomics, these markers are the levels of gene products, such as can be read using in situ hybridization or hybridization chain reaction (HCR).Each region of the sample has a unique feature of marker gene levels, if enough marker genes are used, then it can clearly define the region.Biological samples can be separated and processed by standard single cell methods, and then the measured levels of these marker genes are used to determine the position of cells in biological tissues.

[0005] Such methods require prior information about how these marker genes are distributed in biological tissues. However, mutants (including cancer samples) do not have such available prior information. Therefore, methods that do not require prior information about the distribution of marker genes in target biological tissues have been developed.

[0006] Non-a priori information methods focus on obtaining 2D slices of biological tissue and placing them on slides. For example, to perform spatial transcriptomics, slides are already designed with oligonucleotide markers at known locations, and the first step of RNASeq library preparation is performed on the slides before the biological tissue is digested and mixed. The markers can then be used to determine the origin of each read in the resulting RNASeq data in the biological sample. Alternatively, slides are serially stained with probes for specific combinations of gene products and imaged to determine the levels of these gene products in space.

[0007] However, all of these techniques are inherently two-dimensional (2D) and suffer from drawbacks such as poor capture depth (i.e., the number of unique mRNA reads per cell).

[0008] For example, a publication entitled “Unraveling the Complexity of the Cancer Microenvironment With Multidimensional Genomic and Cytometric Technologies.” (De Vries NatasjaL et al, FRONTIERS IN ONCOLOGY 2020, vol. 10, 2020, page 1254) teaches slicing biological tissues and marking endogenous patterns of undisturbed tissues. Single-cell data are obtained by flow cytometry and mass cytometry, which utilize antibodies coupled to fluorescent dyes or heavy metal isotopes to immunodetect isolated cells. For single-cell RNA sequencing, antibodies coupled to oligonucleotides can be used to simultaneously retrieve information on protein and RNA expression of single cells. This data can then be placed in space by integrating it with spatial omics information.

[0009] The Niche-seq technology is described in a publication titled “Integrating single-cell and spatial transcriptomics toelucidate intercellular tissue dynamics” (Longo Sophia K et al, in Nature Reviews Genetics, vol. 22, no. 10, 18 June 2021).

[0010] The two publications mentioned above propose data from two different techniques: 1) spatial omics and 2) single-cell omics. This means that different cells are collected for each technique. The cells used in the single-cell omics approach need to be deconvolved or mapped onto the cells used in the spatial omics approach. This approach introduces errors because the mapping is not one-to-one and there is no guarantee that the same cell types are collected by both techniques.

[0011] The object of the present invention is to provide a method for obtaining spatial information of biological components in a biological sample without requiring a priori information. Summary of the Invention

[0012] To this end, the present invention proposes a method according to claim 1 and a system according to claim 14 .

[0013] A method for determining the spatial location of one or more biological components in a sample includes: optically encoding the biological components in the sample by a plurality of fluorophores to generate a combination of active fluorophore concentrations that indicates the spatial location of the one or more biological components; separating the biological components from the sample to generate an isolated biological composition of the biological components; and mapping the fluorescence measured from the isolated biological components to the spatial location of the biological components in the unseparated sample.

[0014] Therefore, the present invention proposes a novel method for determining the spatial location of one or more biological components (e.g., cells) within a sample. The present invention proposes optically encoding different components (e.g., cells) of a sample using unique signatures. By measuring and analyzing the fluorescence after separation, the location of the optically encoded components before sample separation can be determined.

[0015] The step of optically encoding biological components in the sample using a plurality of fluorophores to generate a combination of active fluorophore concentrations that indicates the spatial location of one or more biological components is the step of applying a coordinate system to the sample. The active fluorophore concentrations represent spatial coordinates in the coordinate system. Thus, each component of the sample has unique sample coordinates or spatial data in the coordinate system, represented by a combination of active fluorophore concentrations.

[0016] Therefore, for a biological component, the combination of active fluorophore concentrations is a unique signature, and this unique signature can identify the location of the biological component in a sample.

[0017] In other words, a set of active fluorophore concentrations directly represents the spatial location of the biological component. Therefore, the single-cell omics data and spatial data are generated from exactly the same biological components.

[0018] On the other hand, in the present invention, omics data of biological components whose spatial locations in a sample are clearly known can be obtained.

[0019] This is superior to prior art methods, which integrate data from two different technologies: spatial omics and single-cell omics. In the prior art, different cell samples are collected for each technology because once a cell is used for one technology, it cannot be used for the other technology. Therefore, the prior art methods deconvolute or map the cells used in the cell omics technology to the cells used in the spatial omics technology. In the present invention, the spatial information and the omics information of the biological composition are derived from the same biological composition. There is no need to perform any form of mapping from the spatial omics method to the single-cell omics method, thereby avoiding the introduction of errors. In addition, less tissue needs to be used.

[0020] The encoded coordinate system is preferably an orthogonal Cartesian coordinate system.

[0021] In one aspect, the biological sample is chemically treated to render it optically transparent.

[0022] Optical encoding of biological components can include staining a sample with the plurality of fluorophores and, after staining, varying the concentration of active fluorophores for each of the plurality of fluorophores in the sample. By varying the concentration of active fluorophores, a unique signature for each component can be generated. A coordinate system is applied to the sample. For each biological component, the unique signature is a set of sample coordinates in the coordinate system, thereby providing spatial data identifying the location of the biological component in the sample.

[0023] In one aspect, the method comprises generating multiple gradients of active fluorophore concentration in the sample, in particular, wherein the multiple gradients of active fluorophore concentration are orthogonal to each other.Having three orthogonal gradients enables marking three spatial directions in an orthogonal Cartesian coordinate system.

[0024] Changing the concentration of active fluorophores can be done by one of photobleaching, photoactivation, or photoconversion. A gradient that is constant across the sample can be generated.

[0025] The alteration can be effected by photobleaching, photoactivation or photoconversion using a light sheet to which sections of the sample are exposed for varying exposure times.

[0026] On the other hand, before the multiple gradients are generated, the ratio of the fluorophore concentrations on the biological object is constant between the compositions.

[0027] In one embodiment of the present invention, it is envisaged that at least one, preferably two, of a plurality of fluorophores are covalently attached to the oligonucleotide.

[0028] Optical encoding can be performed using one or more different fluorophores, where different fluorophores have different excitation, activation, or conversion spectra. Different emission spectra define independent fluorescence channels.

[0029] Additional fluorophores can be used as control fluorophores. The control fluorophore is maintained constant while the other fluorophores are varied to optically encode biological components in the sample. A step can be provided to normalize the measured fluorescence level of the varied fluorophore relative to the fluorescence level of the control fluorophore. Using a control fluorophore can help improve the accuracy of the assay, particularly if the initial staining in the sample is uneven before the fluorophore is varied.

[0030] The method can include using a calibration sample, wherein the concentration change of each active fluorophore is recorded by imaging the calibration sample as each fluorophore changes with increasing exposure in different tissue sections. A modification parameter can be determined for modifying the sample from the resulting modified calibration map to determine the exposure at each position along the axis to achieve a desired gradient in the sample.

[0031] The method may include mapping measured fluorescence levels of different fluorophores of the separated biological constituents to physical space in the sample before separation, such that each color combination measured after separation is assigned to a possible location in the physical space.

[0032] On the one hand, mapping the measured fluorescence levels of the separated biological components to a physical space includes: imaging the biological sample before separation, and mapping the measured color space of the separated biological components to the measured color space in the system used to image the biological sample before separation through a histogram matching algorithm for each fluorescence channel.

[0033] Preferably, single fluorophore stained control samples are used to determine the crosstalk between pairwise combinations of channels in the system used to image the sample before separation and the system used to measure the signal of the biological composition after separation, so as to correct the channel data in the two systems before performing histogram matching for each channel between the two instruments.

[0034] The sample can be reconstructed from the measured fluorescence.

[0035] The present invention also provides a system for determining the spatial location of one or more biological components in a sample. The system includes: an optical encoding unit configured to optically encode the biological components in the sample using multiple fluorophores to generate a combination of active fluorophore concentrations that indicates the spatial location of the one or more biological components; an optical measurement system for measuring fluorescence after separation, including a fluorescence-activated cell sorter or analyzer, or a microfluidic device; and a mapping unit configured to map the fluorescence measured from the separated biological components to the spatial location of the biological components in the unseparated sample.

[0036] Biological constituents in a sample can be optically encoded by staining and altering them with multiple fluorophores to generate a combination of active fluorophore concentrations that indicates the spatial location of one or more biological constituents.

[0037] The optical encoding unit may include an imaging unit to change the spatial distribution of the concentrations of the multiple fluorophores and measure the spatial distribution of the concentrations of the multiple fluorophores after the change, specifically, wherein the imaging unit includes a selective plane illumination microscope for imaging the sample before separation.

[0038] Therefore, this paper describes a method with potentially equal resolution in all dimensions. Attempts to perform 3D omics approaches via slide-based methods have various problems / issues, including aligning consecutive slides, loss of spatial resolution and information in cut edges, varying resolution along different axes, labor time, and cost.

[0039] The method proposed in this paper does not require the integration of spatial omics data with scRNASeq data, thus reducing the risk of errors because they are not caused by inaccurate mapping of single-cell RNASeq data to spatial information.

[0040] This approach potentially enables a greater depth of capture than slide-based methods. In fact, the method and system can be used like a module in a method workflow. The method can be performed directly upstream of single-cell RNASeq, which means the capture depth approaches that of scRNASeq. This is much higher than the capture depth of existing slide-based spatial omics methods, which require single-cell RNA sequencing data to supplement spatial information from spatial omics methods to achieve equivalent capture depth.

[0041] Finally, this approach achieves the simultaneous acquisition of the spatial information of spatial omics methods and the capture depth of single-cell RNA-Seq in a single technique. Therefore, less biological tissue is required, which is particularly important in situations where tissue is scarce, such as tumor biopsies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A general schematic diagram of a system according to one aspect of the present invention is shown.

[0043] Figure 2 A general schematic diagram of a method according to one aspect of the present invention is shown.

[0044] Figure 3 A method of optical encoding according to one aspect of the present invention is shown.

[0045] Figure 4 A method of optical decoding according to one aspect of the present invention is shown.

[0046] Figure 5 An example is shown. DETAILED DESCRIPTION

[0047] The present invention will now be described with reference to the accompanying drawings. It should be understood that the embodiments and aspects of the present invention described herein are merely illustrative and do not in any way limit the scope of protection of the claims. The present invention is defined by the claims and their equivalents. It should be understood that features of one aspect or embodiment of the present invention may be combined with features of different aspects and / or embodiments of the present invention.

[0048] Figure 1 FIG. 1 shows an overall schematic diagram of a system 1 for determining the spatial location of multiple biological components in a biological sample 10. Figure 2 An overall schematic diagram of a method for determining the spatial location of biological constituents in a biological sample 10 is shown.

[0049] A biological sample 10 comprises a biological component 12 and information about the spatial distribution of the biological component 12 in the sample 10 is desired.

[0050] In the present invention, an example of the biological composition 12 is the cells of the biological sample 10 .

[0051] The method includes the step of optically encoding (S100) biological components 12 in a biological sample 10 to generate a unique optical signature for each biological component 12 in the biological sample 10. In one aspect, the biological components 12 in the biological sample 10 are optically encoded by a plurality of fluorophores 31, 32, 33 by generating a combination of active fluorophore concentrations that indicates the spatial location of the biological components in the biological sample 10. A unique signature indicating the spatial location is obtained by the combination of different active fluorophores, and the unique signature is used as the optical signature.

[0052] Each biological component of the biological sample 10 has a set of sample coordinates or spatial data represented by a combination of active fluorophore concentrations. The set of sample coordinates is an optical feature.

[0053] Optical encoding will be referred to later Figure 3 Describe in more detail.

[0054] After optical encoding, the method for determining the spatial location of the biological composition 12 in the biological sample 10 comprises a step of separating S200 the biological composition 12 from the biological sample 10 to generate a separated biological composition 12 of the biological composition 12 .

[0055] After separation, in step S320, the measured fluorescence from the separated biological components 12 is mapped to the spatial position of the biological components 12 in the unseparated sample 10 to obtain the original position of the biological components in the unseparated sample 10. The mapping will be referred to later. Figure 4 Provide a description.

[0056] Measuring the fluorescence of a fluorophore from an isolated biological component 12 (here, isolated cells) is equivalent to reading a unique signature of the biological component. Therefore, the measurement can provide information about the spatial location of the cells before isolation. The method then includes mapping the measured optical signature of the isolated biological component 12 to the spatial location of the cells in the unisolated sample 10.

[0057] The concentration of a set of active fluorophores directly represents the spatial location of biological components in a biological sample. Therefore, single-cell omics data and spatial data are generated from exactly the same biological components.

[0058] It will be appreciated by those skilled in the art that the present invention does not suffer from the drawbacks of known 3D imaging methods using slide-based methods, such as problems with aligning consecutive slides, loss of spatial resolution and information in cut edges, different resolutions along different axes, labor time and cost.

[0059] Now refer to Figure 3 Explain optical encoding in detail.

[0060] First, a biological sample 10 is prepared. The preparation includes fixing and permeabilizing cells (as an example of biological components) of the biological sample 10 using a fixative and a permeabilizing agent in step S110.

[0061] Suitable fixatives include, but are not limited to, paraformaldehyde / formalin, methanol, acetone, DSP and its derivatives, and flash freezing.

[0062] The sample 10 can be permeabilized by a permeabilizing agent. Suitable permeabilizing agents include, but are not limited to, common organic solvents and detergents, such as Triton X-100, Tween-12, CHAPS, sodium lauroyl sarcosinate, DMSO, SDS, and saponin. Some of these agents can simultaneously fix and permeabilize the sample 10.

[0063] Next, in step S120, the biological sample 10 is stained by a plurality of fluorophores 31, 32, 33, and 34. The staining is performed by at least three different fluorophores 31, 32, and 33. The three different fluorophores 31, 32, and 33 have different excitation spectra and different emission spectra.

[0064] In the detailed example, three different fluorophores are used to generate a coordinate system having three dimensions. However, this is not limiting of the present invention and other coordinate systems are contemplated.

[0065] The different fluorophores include a first plurality of first fluorophores 31 having a first excitation spectrum and a first emission spectrum, a second plurality of second fluorophores 32 having a second excitation spectrum and a second emission spectrum, and a third plurality of third fluorophores 33 having a third excitation spectrum and a third emission spectrum.

[0066] The fourth fluorophore 34 also serves as a control fluorophore. The four different fluorophore concentrations are stoichiometric, i.e., the fluorophore concentrations are proportional to one another. Thus, the coded fluorophore concentrations 31, 32, 33 can be normalized by the fourth (control) fluorophore 34. The use of a control fluorophore or control channel relaxes the requirement for a completely spatially uniform fluorophore concentration. Thus, the fourth fluorophore 34 is a staining control that is used to normalize the signals from the other three fluorophores and enables control for different staining efficiencies in different parts of the sample 10.

[0067] The initial fluorophore concentration of all fluorophores 31 , 32 , 33 , 34 should be as uniform as possible throughout the sample 10 in order to obtain a well-defined fluorophore concentration gradient.

[0068] Suitable fluorophores include, for example, cell marker fluorophores, such as those that bind to genomic DNA or RNA, those that bind to cell membranes, and those that bind to any other component of the cell, those that are covalently linked directly to the oligonucleotide or indirectly through a probe-involving process. Such probes include, but are not limited to, padlock probes, snail probes, hybridization chain reactions, or nanospheres generated by rolling circle amplification.

[0069] If the stain is an oligonucleotide or probe that binds directly or indirectly to genomic DNA, these fluorophores (31, 32, 33, 34) can be covalently linked to oligonucleotides whose sequences appear multiple times in the genomes of multiple species.

[0070] If the stain is an oligonucleotide or probe that binds directly or indirectly to RNA, these fluorophores (31, 32, 33, 34) can be covalently linked to the oligonucleotide or probe whose sequence may be present multiple times in the expressed RNA.

[0071] Suitable hybridization mixtures for staining biological objects 10 include, but are not limited to, DMSO, Triton X-100, Tween-12, sodium citrate saline, dextran sulfate, Herapin, SDS, formamide, Denhardt's solution, and EDTA.

[0072] If the stain is an oligonucleotide, the biological sample 10 may be heated and cooled one or more times to promote annealing.

[0073] In one aspect, two oligonucleotides with identical sequences are envisioned, each having two fluorophores covalently attached.

[0074] The fluorophores 31 , 32, 33, 34 are positioned as far apart as possible on the oligonucleotide and / or as spectrally different as practical to avoid any non-radiative energy transfer.

[0075] It should be noted that, in the case where the fluorophores 31, 32, 33, 34 are covalently linked directly or indirectly to oligonucleotides, one or more of the fluorophores 31, 32, 33, or 34 can be linked to any given oligonucleotide. In the case where the fluorophores 31, 32, 33, 34 are linked to the same oligonucleotide, optimization techniques involve maximizing the physical and spectral distances between the fluorophores 31, 32, 33, 34. In the case where the fluorophores 31, 32, 33, 34 are linked directly or indirectly to oligonucleotides, the sequences of these oligonucleotides should be found in the genome of the species of the biological object 10 being stained.

[0076] In the example where the oligonucleotides are covalently linked to fluorophores or probes, they may be annealed to the genomic DNA or RNA of the biological constituents 12 of the biological sample 10 by heating and cooling steps.

[0077] It should be noted that after staining and before changing the fluorophore concentration, the biological sample 10 can be washed to remove unbound dye and post-fixed. Suitable cleaning agents include, but are not limited to, PBS, TBS, maleic acid buffer, and HBSS. Suitable post-fixatives include, but are not limited to, paraformaldehyde / formalin, methanol, acetone, DSP, and its derivatives. Washing the biological sample 10 after staining can improve the signal-to-noise ratio during subsequent method steps.

[0078] The next step is to prepare the biological sample 10 for the optical encoding process. This may include mounting the sample 10, possibly dehydrating it for a specific clearing protocol, and chemically clearing the sample 10. It is often convenient to mount the sample 10 in a hydrogel, which provides protection and mechanical support during processing. Organic solvent-based clearing protocols, such as BABB and the DISCO series, require dehydration of the sample 10 prior to immersion in an organic solvent for clearing.

[0079] Suitable mounting media include, but are not limited to, low melting point agarose, agarose, polyacrylamide, and Matrigel.

[0080] Suitable dehydrating agents include, but are not limited to, methanol, ethanol, propanol, and tetrahydrofuran (THF).

[0081] Suitable clearing agents and methods include, but are not limited to, BABB, CLARITY, PACT, PARS, 3DISCO, iDISCO, Spalteholz, glycerol, and CUBIC. For simplicity, in the remainder of the detailed description, clearing using the BABB protocol is described, although other clearing protocols may be used.

[0082] The next step in the process is to image and vary the active concentrations of the fluorophores 31, 32, 33, 34 at step S130, thereby generating multiple gradients of active fluorophore concentrations in the biological sample 10. Step S130 can be performed using an optical encoding system for varying and / or imaging the distribution of the active fluorophores 50.

[0083] There are a variety of mechanisms that can be used to optically alter the concentration of active fluorophores. Depending on the choice of fluorophore 31, 32, 33, 34 used, these mechanisms include, but are not limited to, photobleaching, photoconversion, or photoactivation. For simplicity, the detailed description of the present invention will describe the use of photobleaching.

[0084] One method of performing optical encoding S100 is to use an optical instrument that employs scanning with spatially non-uniform illumination. The desired distribution of active fluorophores in sample 10 results from the specific non-uniform illumination and scanning pattern employed. Examples of such instruments include, but are not limited to, light sheet microscopes, confocal microscopes, or two-photon microscopes. For simplicity, in the remainder of the detailed description, the imaging and alteration (i.e., optical encoding) of step S130 is described as being performed using a SPIM microscope.

[0085] An alternative to using scanning instruments for optical encoding is illumination with a collimated beam with an appropriate intensity profile. For example, a beam with a logarithmic intensity profile can be used to bleach a linear gradient along one dimension of a uniformly stained sample without scanning.

[0086] After staining S120 , a plurality of gradients are generated by varying S130 the active fluorophore concentration of each of the plurality of fluorophores 31 , 32 , 33 , 34 in the biological sample 10 .

[0087] Optimally, the multiple gradients of active fluorophore concentrations are orthogonal to one another. A first gradient is for a first fluorophore having a first excitation spectrum and a first emission spectrum, a second gradient is for a second fluorophore having a second excitation spectrum and a second emission spectrum, and a third gradient is for a third fluorophore having a third excitation spectrum and a third emission spectrum.

[0088] In other words, each cell can be identified by a unique optical signature represented by three fluorophores, one for each dimension.

[0089] It will be appreciated that a minimum of three fluorophores is required to unambiguously encode each of the three spatial dimensions. The optical signature is a set of sample coordinates represented by the three fluorophores, one for each dimension. Coordinate systems with other spatial dimensions can be considered.

[0090] Calibration samples were first imaged in 3D in all 4 channels and successful staining was confirmed.

[0091] The concentration of the active fluorophores 31, 32, 33, 34 can then be altered by photoactivation, photoconversion, or photobleaching, depending on the type of fluorophores used 31, 32, 33, 34. The resulting concentration profile of the active fluorophores 31, 32, 33, 34 can include any that provides unambiguous positional information for one or more cells within the biological sample 10.

[0092] In one aspect, cells are imaged using a selective plane illumination microscope (SPIM) or other light sheet microscope 50. The calibration sample is bleached in a single plane, i.e., in a different non-overlapping plane in a different part of the calibration sample for each channel or each channel to be bleached.

[0093] By imaging a biological sample 10, the decrease in the concentration of active fluorophores 31, 32, 33, 34 is recorded as the sample bleachs over time. For each fluorophore 31, 32, 33, 34, a bleaching calibration map 48 is generated that determines the bleaching parameters required to bleach to a desired level of active fluorophore concentration. The bleaching parameters for each of the three channels are read from the calibration map generated for each fluorophore 31, 32, 33, 34 in the bleaching calibration sample. The bleaching calibration map 48 can be used to determine the exposure at each position along the axis to achieve the desired gradient in the biological sample 10.

[0094] Light-sheet microscopy 50 can rapidly bleach a given plane within a biological sample 10 while leaving the rest relatively unaffected. In the case of light-sheet microscopy, the light sheet can be generated using a cylindrical lens or a rapidly translating laser line (e.g., using a galvo mirror or resonant scanner). Furthermore, light-sheet microscopy can easily generate 3D images of the resulting bleaching pattern, which can be used to confirm the observed distribution of active fluorophores.

[0095] Post-bleaching imaging of the biological sample 10 can be used to map fluorescence measured from the isolated biological components 12 to the spatial location of the biological components 12 in the unisolated sample, as described later in the present invention. Post-bleaching imaging of the biological sample 10 can be used for mapping in conjunction with a single-color control that determines the color space mapping between the imaging / modification system and the isolated cell detection.

[0096] As mentioned above, it is desirable to generate three orthogonal gradients, i.e., orthogonal monotonic distributions, for example. In practice, linear distributions tend to optimize the signal-to-noise ratio and make the positional accuracy spatially uniform. After the desired pattern is written into the fluorophore, the pattern is imaged in 3D to quantitatively record the actual pattern achieved.

[0097] Before changing the intensity of the fluorophore, the Pearson correlation coefficient of each changed fluorophore 31, 32, 33 relative to the used control fluorophore 34 should be sufficiently high, where each point in the correlation analysis is the intensity of the fluorophore of a single cell. For example, the Pearson correlation coefficient is preferably greater than 0.2, more preferably greater than 0.5, and even more preferably at least 0.9.

[0098] One technique for implementing the step of varying the concentration of S130 active fluorophores is photobleaching using a light sheet 40 to which sections of the biological sample (10) are exposed with varying exposure times.

[0099] The bleaching parameters for each of the three channels were derived from a calibration plot generated by bleaching each fluorophore in the calibration samples.

[0100] One approach consists of generating a linear distribution of active fluorophores, which should achieve uniform resolution along the axis. The resulting gradient is linear rather than exponentially decaying, so that the position error is uniform at any position along the axis. If an exponentially decaying gradient is generated, for example by varying the exposure linearly along the axis, then different amounts of position error along the axis are obtained depending on the gradient of the curve.

[0101] The first dimension x is bleached using a light sheet perpendicular to the x dimension, which excites one of the fluorophores 31 that resides in the biological sample 10 for a different time.

[0102] The longer the light sheet is held at each location, the lower the resulting concentration of unbleached fluorophores in the corresponding plane. Alternatively, different regions of the biological sample 10 can be scanned a different number of times to bleach the gradient, or the laser power can be varied spatially. Multiple scans are effectively the same as simply varying the exposure.

[0103] The second dimension y is bleached by rotating the sample 90 degrees about the vertical axis (z) and then repeating the process used for bleaching the x dimension with a laser line appropriate for the second fluorophore 32 .

[0104] The third dimension z is bleached using a light sheet of finite height with a laser line tailored to the third fluorophore 33, which is scanned for equal time across the biological sample 10. Between scans, the sample 10 is gradually raised (translated along the z axis) out of the path of the light sheet, so that different areas in the sample are illuminated different times, and a gradient of active fluorophores 33 is bleached along the third dimension.

[0105] As an alternative to the process described in the previous paragraph, to bleach the z dimension, the sample 10 can be rotated 90 degrees about the horizontal axis and then the process for y-dimension bleaching can be repeated using a laser line suitable for the third fluorophore 33 .

[0106] As an alternative to the procedure described in the previous paragraph, which assumes sequential bleaching of the sample 10 along three axes using a SPIM instrument with illumination along a single axis, an instrument can be designed that can illuminate along two or more axes. This allows bleaching along multiple axes, either sequentially or simultaneously, without the need to rotate the sample between bleachings.

[0107] The result is a biological sample 10 having orthogonal active fluorophore gradients along three spatial dimensions, plus an unbleached control fluorophore 34 .

[0108] After photobleaching, the concentration distribution of the four fluorophores is determined by 3D imaging of the biological sample 10 in all four channels.

[0109] This is not the only method, the change can be made by either photoactivation or photoconversion.

[0110] The next step is to make the sample "opaque" and return it to the state before transparency. For those schemes that need dehydration, this can be carried out by reversing the transparency scheme and possibly rehydration. Suitable transparency agents include but are not limited to methanol, ethanol and propanol. Suitable rehydration agents include but are not limited to PBS, TBS, maleic acid buffer and HBSS.

[0111] The next step includes separating S200 the biological composition 12 from the biological sample 10 to generate a separate biological composition 12 of the biological composition 12 .

[0112] The sample 10 can be enzymatically separated using enzymes including, but not limited to, collagenase, dispase, and trypsin. Tissues can also be mechanically separated by vortexing, ultrasound, manual or electric grinding (pestel), or using a specialized tissue dissociator (e.g., gentleMACS). A combination of enzymatic and mechanical methods can also be used to separate the biological sample 10.

[0113] The next step involves separating the biological components 12 into separate compartments for downstream processing. The biological components can be separated by FACS instrumentation, microfluidic devices, or dilution. Compartments include wells in multiwell plates, fabricated microwells, test tubes, spots on glass slides, and bubbles in water-in-oil emulsions.

[0114] After separation, the next step is optical decoding S300, which involves measuring S310 the fluorescence of the separated biological components and mapping S320 these fluorescence measurements to the spatial locations of the biological components in the unseparated sample.

[0115] The following will refer to Figure 4 Provide explanation.

[0116] First, fluorescence is read from the separated biological component 12. More precisely, during or after separation of the components, the fluorescence intensity of the various fluorophores should be read to determine the location of origin of the biological component 12 in the sample 10.

[0117] To read the intensity of the fluorophores 31, 32, 33, 34, a measurement system (80) can be used, which includes an excitation system 60 (e.g., a laser, LED, or fluorescent lamp) to excite the fluorophores, and a detection system 70 (e.g., a camera, PMT, or other type of photodetector of the optical decoding system 300) to measure the fluorescence. The detection (step S310) can be performed in the context of a fluorescence excitation / detection device built into a fluorescence activated cell sorter (FACS) instrument, a fluorescence plate or slide reader, or a microfluidic device.

[0118] Then, any omics analysis can be performed on the biological components 12 in each compartment, and the location of each component 12 in the original biological sample 10 can be derived from the fluorescence levels of the three axes normalized to the control channel.

[0119] At step S320, the measured fluorescence levels are mapped to the spatial locations of the unseparated cells in the biological sample 10. Each color combination measured after separation is thereby assigned to a unique location in the physical space of the biological sample 10. In effect, the set of active fluorophore concentrations directly represents the spatial location of the biological constituents in the biological sample.

[0120] For each biological component 12, the levels recorded in the three bleached channels F1, F2, F3 can be normalized by the control non-bleached channel F4. Normalization means dividing the levels of the bleached channels F1, F2, F3 by the level of the control channel F4.

[0121] Typically, a linear distribution of active fluorophores can be used so that the resulting optical signature directly represents the original position of the biological component 12 in the biological sample (10). The mapping between the normalized "color code" of a cell (F1 / F4, F2 / F4, F3 / F4) and the original position x, y, z of the cell in the biological sample 10 is a simple set of linear equations.

[0122] In other words, the original 3D position of the biological composition 12 is reconstructed by normalizing the active fluorophore level of each altered bleached, photoactivated, or switched channel by the active fluorophore level of the unaltered control channel to produce an optical signature for each composition 12. The resulting optical signature represents the position in physical space, i.e., the original position {x, y, z} of each composition 12 in the biological sample 10. This approach can be performed in linear algebraic terms if the distribution of active fluorophore concentrations forms the basis for the optical encoding.

[0123] Optical encoding imposes a coordinate system on the biological sample, and for the biological constituents, the concentration of the active fluorophore is a set of spatial coordinates in said coordinate system.

[0124] Before separation, the color code distributions or values ​​(I1, I2, I3, and I4) can be measured in a light sheet microscope for photobleaching 50. As described above, this mapping from "color space" to "physical space" is linear for orthogonal linear distributions of active fluorophores.

[0125] After separation, the fluorescence levels (F1, F2, F3, and F4) in the separated cells can be recorded, for example, by measurement by a detection system 70 (eg, a FACS instrument).

[0126] Ideally, the system used to image the biological sample 10 before separation but after altering the distribution of the active fluorophore (e.g., a light sheet microscope 50) and the detection system 70 used to detect fluorescence levels in the separated cells (e.g., a FACS instrument) respond identically to a given concentration of the active fluorophore.

[0127] Those skilled in the art will appreciate that even if one attempts to closely match the responses measured by the optical encoding system used to alter and / or image the distribution of active fluorophores and the system used to read fluorescence after separation of the biological composition 12, the responses will not be identical. For example, if the optical encoding system used to alter and / or image the distribution of active fluorophores is a light-sheet microscope, and the system used to read fluorescence after separation of the biological composition 12 is a detector system 70, similar laser lines can be used for excitation in the light-sheet microscope 50 and the detector system 70 to closely match the responses. However, the responses will not be identical. This is due to, for example, slightly different lasers, completely different detectors, different filters, and different sample media.

[0128] Therefore, it is proposed to map the fluorescence {F1 / F4, F2 / F4, F3 / F4} recorded after separating the color space to the fluorescence {I1 / I4, I2 / I4, I3 / I4} recorded before separating the color space, which can then be mapped to the physical space {x, y, z} as described above.

[0129] Therefore, mapping the measured color space of the isolated cells to the physical space, i.e., mapping {F1, F2, F3, F4}→{x, y, z}, first involves mapping the measured color space of the isolated cells to the imaging color space in the imaging system (50) used to image the sample (10), i.e., mapping {F1, F2, F3, F4}→{I1, I2, I3, I4}. The mapping from the imaging color space to the physical space {I1 / I4, I2 / I4, I3 / I4}→{x, y, z} is very simple because one has a voxel dataset from the post-bleaching sample scan: in this voxel data, the voxel coordinates represent the physical space {x, y, z} and the voxel values ​​represent the imaging color space {I1 / I4, I2 / I4, I3 / I4}. This mapping from the measured color space of the isolated cells to the physical space is performed by calibrating the responses of the system 50 for imaging the distribution of the active fluorophores and the system 80 for reading the fluorescence after separation of the biological component 12, using four calibration samples, each containing only one of the four fluorophores 31, 32, 33, 34. The mapping between the response curves of the imaging system 50 and the detection system 80 gives the desired relationship between the fluorescence {F1, F2, F3, F4} recorded after separation of the color space and the fluorescence {I1, I2, I3, I4} recorded before separation of the color space, and thus maps to the physical space coordinates {x, y, z}, i.e., {F1, F2, F3, F4} → {I1, I2, I3, I4} → {I1 / I4, I2 / I4, I3 / I4} → {x, y, z}. Suitable methods for this mapping include, but are not limited to, histogram matching algorithms for each channel or the use of artificial intelligence methods.

[0130] Histogram matching is performed using fluorescence signals measured in all four channels of four biological samples, each stained with only one type of fluorophore, for a system for imaging changes in and / or the distribution of active fluorophores and a system for reading fluorescence after separation of biological components 12. The measured optical signature of each biological component 12 after separation is then mapped to the corresponding optical signature in the system for 3D imaging the fluorophore distribution before separation.

[0131] From the resulting adjusted optical signature, the physical location of the biological component 12 before separation can be read out by comparing the optical signature with the fluorophore concentration at each physical location in the 3D image (after modifying the distribution of active fluorophores) using a distance metric such as Euclidean distance and selecting the most likely location where the metric is minimized. Other suitable distance metrics for such mapping include, but are not limited to, Pearson, Kendall's tau, Manhattan, and Spearman rank correlation distances.

[0132] There may be situations where the optical signature cannot be encoded as a monotonic gradient, but the optical signature can still be used in some other way to generate unambiguous positional information. For example, if more than four suitable fluorophores are available, two of the fluorophores can be combined along an axis to make the localization unambiguous, even if no fluorophore alone provides unambiguous information. Then, by comparing the set of normalized optical signatures of the biological composition 12 with the optical signature encoded in the sample 10 (read during the post-bleaching imaging phase) using any form of suitable distance metric, such as Euclidean distance, a mapping step can be used that maps the resulting biological composition 12 to its most likely location of origin in the sample 10. Other suitable distance metrics for such mapping include, but are not limited to, Pearson, Kendall's tau, Manhattan, and Spearman rank correlation distances.

[0133] If enough biological components 12 are mapped to their expected locations of origin in the biological sample 10, the original tissue can be reconstructed (S400).

[0134] Figure 5 An example of data obtained by the methods and systems of the present invention is given, and the protocol is described below.

[0135] Imaging and photobleaching Samples were imaged and bleached on a custom-made cylindrical lens-based OPTiSPIM light sheet microscopy microscope. Briefly, 50 mW 405 nm, 50 mW 488 nm, and 50 mW 639 nm lasers were used for imaging and bleaching of Alexa 405, FAM, and Alexa 647 fluorophores, respectively. A 5 mW 543 nm laser was used to image the internally stained control TAMRA fluorophore. Bandpass filters of 447 BP60, 525 BP50, 585 BP60, and 700 BP75 were used for detection of Alexa 405, FAM, TAMRA, and Alexa 647 fluorophores, respectively. A 2.5 × N Plan air objective (Leica, NPLAN, NA = 0.07) was used to illuminate the sample. A 5× NPlan Epi air objective (Leica, NPLAN EPI, NA=0.12, WD=14 mm) and a 12-bit cooled Hamamatsu ORCA-ER C4742-80 CCD camera were used for detection.

[0136] Calibration samples are used to calibrate for photobleaching by placing the light sheet at fixed positions for each laser line and monitoring the decrease in fluorescence signal intensity with exposure time. By reading these calibration plots (one for each fluorophore / laser line), the photobleaching exposure time for different positions of the light sheet in the test sample is determined.

[0137] Handling bleached samples After bleaching, sample 10 was subjected to one quick wash and two longer washes (>4 hours) in methanol. Sample 10 was then removed from the LMP agarose and rehydrated by one quick wash and two longer washes (>4 hours) in PBS. For experiments in which the limb ectoderm and mesenchyme were analyzed separately, the limb buds were incubated in 0.5% trypsin-EDTA 10x (without phenol red - Gibco 15400-054) at room temperature for 15 minutes, and the ectoderm was removed from the limb with forceps. For all other experiments, the tissue was digested with 0.22 μm filtered 10 mg / ml collagenase / dispase (containing 1 / 1000000 Triton) in PBS at 37°C and 600 RPM for 2 hours. The biological sample 10 was mechanically agitated regularly using a gilson pipette. After tissue separation, the sample 10 was centrifuged at 600g for 5 minutes at room temperature. The cell pellet was resuspended in 0.5-1 ml of PBS, filtered through a 40 µm filter, and then processed on a FACS analyzer (Fortessa). It should be noted that this is not a limiting example and other equipment, such as a cell sorter (FACSAria), can be used.

[0138] Separated detector system In the example shown, the fluorescence level of cell is simply analyzed by Fortessa FACS analyzer.Use the FACS gating for side scatter area and forward scatter to remove cell debris (gate P1).Then singlet is selected highly compared to area gating (gate P2) by forward scattering.Use PE-A (corresponding to 586BP15 bandpass filter to detect TAMRA staining control) to select fluorescent cells and remove any residual cell debris (gate P3) compared to forward scattering gating.

[0139] The fluorescence channel levels of PACB (corresponding to a 450BP50 bandpass filter to detect Alexa405), FITC-A (corresponding to a 530BP28 bandpass filter to detect FAM), and APC-A (corresponding to a 670BP14 bandpass filter to detect Alexa647) were normalized to the fluorescence channel levels of PE-A (to detect TAMRA) to generate Figure 5 Therefore, for simplicity, in this example the reconstruction assumes the same responses from the FACS and SPIM instruments, and the color space is assumed to directly represent the physical space (as described in paragraphs 104-107) since linear gradients are generated in all dimensions.

[0140] Figure 5 The results of a 3D reconstruction of an E10.5 hind limb bud from a mouse embryo that had undergone the C3PO process are shown. It is known that pre-detached ectodermal cells form a cap-shaped structure on the mesenchyme with a central depression and a thickened, curved edge that corresponds to the well-characterized apical ectodermal ridge (AER). Figure 5 The upper left figure shows that ectodermal cells are indeed found to have a curved thickening area, precisely located near the position where the AER is expected to be found (low x dimension). The upper right figure shows a transverse plate-like structure of ectodermal cells, with the center of the ring of ectodermal cells having a distinct depression, consistent with expectations. Very few ectodermal cells are detected on the straight edge side of the limb bud (high x dimension). This is where the limb bud is separated from the rest of the embryo, so there should be no ectoderm along this edge, which is exactly the same as what is seen in the reconstruction. Finally, in the figure below, limb bud ectodermal cells (large dots) and limb bud mesenchymal cells (small dots) are shown. Ectodermal cells should surround the mesenchyme because this layer is directly located on the mesenchyme in the wild-type limb bud, which is exactly the structure observed in the reconstruction. In short, the reconstructed limb bud structure is very similar to the structure before separation, which proves that the method described in the present invention is effective.

[0141] Reference Signs List 1 System 10 Biological samples, examples include but are not limited to organs, embryos or biopsy samples 12 Biological components, examples include but are not limited to cells or cell clusters 31, 32, 33 Fluorophores 34 Control fluorophore 40 light sheets 48 Calibration Chart 50. System for modifying and / or imaging the distribution of active fluorophores 60 Excitation System 70 Detection System 80 Optical measurement system for reading fluorescence after cell separation 90 mapping units 100 optical encoding units 300 Optical Decoding System {F1, F2, F3, F4} Fluorescence recorded using the color space separated by (70) {I1, I2, I3, I4} Fluorescence recorded in color space before separation using (50) {x, y, z} physical space

Claims

1. A method for determining the spatial location of one or more biological components (12) in a biological sample (10), the method comprising: - optically encoding (S100) a biological component (12) in a biological sample (10) by a plurality of fluorophores (31, 32, 33, 34) to generate a combination of active fluorophore concentrations within the biological sample (10) that indicates the spatial location of one or more biological components (12); - separating (S200) the biological composition (12) from the biological sample (10) to generate a separated biological composition (12) of the biological composition (12); and - Optical decoding (S300) is performed by measuring (S310) the fluorescence of the separated biological component (12) and mapping (S320) to the spatial position of the biological component (12) in the unseparated sample (10).

2. The method of claim 1 , wherein the step of optically encoding the biological composition comprises: A coordinate system, preferably an orthogonal Cartesian coordinate system, is optically encoded on the biological sample, and the combination of active fluorophore concentrations for the biological component is a set of coordinates in said coordinate system.

3. The method according to claim 2, wherein the step of optically decoding comprises: For each separated biological component, a set of coordinates in the sample coordinate system is determined to obtain the spatial position of each biological component in the unseparated sample (10).

4. The method according to any one of claims 1 to 3, wherein generating a spatial combination of active fluorophore concentrations comprises: The biological sample (10) is stained (S120) by the plurality of fluorophores (31, 32, 33, 34).

5. The method according to any one of claims 1 to 4, wherein the plurality of different fluorophores are attached to a single molecule, such as covalently attached to an oligonucleotide, in particular wherein the plurality of different fluorophores are attached to the part of the biological composition in a constant ratio.

6. The method according to claim 4 or 5, further comprising changing (S130) the active fluorophore concentration of each of the plurality of fluorophores (31, 32, 33, 34) in the biological sample (10) after staining (S120).

7. The method according to any one of claims 1 to 6, comprising generating a plurality of gradients of active fluorophore concentration in the biological sample (10), in particular a plurality of gradients of active fluorophore concentration that are orthogonal to one another.

8. The method according to claim 6 or 7, wherein the change (S130) of the concentration of active fluorophores is implemented by one of photobleaching, photoactivation or photoconversion, in particular, wherein the change (S212) is implemented by photobleaching using a light sheet (40), and areas of the biological sample (10) are exposed to the light sheet (40) with different exposure times.

9. A method according to claim 8, comprising measuring the fluorescence level of the calibration sample before and after varying the concentration of the active fluorophore to generate a bleaching calibration graph (48) which defines the bleaching parameters to be used during varying the resulting level of active fluorophore concentration in the target sample (10).

10. The method according to any of the preceding claims, wherein the optical encoding (S100) is performed by means of different fluorophores (31, 32, 33) having different excitation spectra and different emission spectra defining at least three fluorescence channels.

11. The method according to any of the preceding claims, comprising using an additional fluorophore (34) as a control fluorophore, in particular the method further comprising normalizing the measured fluorescence levels of the non-control fluorophores (31, 32, 33) relative to the fluorescence level of the control fluorophore (34).

12. A method according to any of the preceding claims, comprising mapping the measured fluorescence levels of different fluorophores (31, 32, 33) of the separated biological components (12) to a physical space in the biological sample (10) before separation, such that each color combination measured after separation is assigned to a possible position in the physical space.

13. The method of claim 11, wherein mapping the measured fluorescence levels of the isolated biological components (12) to a physical space comprises: The biological sample (10) is imaged before separation, and the measured color space of the separated biological components is mapped to the color space measured in the system used to image the biological sample (10) before separation, such as by a histogram matching algorithm for each fluorescent channel.

14. The method of claim 12 or 13, wherein a control sample stained with a single fluorophore is used to determine the crosstalk between pairwise combinations of channels in a system for imaging the biological sample (10) before separation and in a system for measuring signals of the biological composition (12) after separation, so as to correct the channel data in the two systems before performing histogram matching for each channel between the two instruments.

15. The method according to any of the preceding claims, further comprising reconstructing the biological sample (10) from the measured fluorescence.

16. A system for determining the spatial location of one or more biological components (12) in a biological sample (10), comprising: - an optical encoding unit (50) configured to optically encode a biological component (12) in a biological sample (10) by means of a plurality of fluorophores (31, 32, 33, 34) to generate a combination of active fluorophore concentrations indicative of a spatial location of one or more biological components (12); - an optical measurement system (80) for measuring fluorescence after separation, comprising a fluorescence activated cell sorter or analyzer, or a microfluidic device; - A mapping unit (90) configured to map the fluorescence measured from the separated biological component (12) to the spatial position of the biological component (12) in the unseparated sample (10).

17. The system of claim 16, wherein the optical encoding unit comprises an imaging unit to change the spatial distribution of the concentrations of the plurality of fluorophores and measure the changed spatial distribution of the concentrations of the plurality of fluorophores, in particular, wherein the imaging unit comprises a selective plane illumination microscope or other light sheet microscope (50) for imaging the biological sample (10) before separation.

18. The system according to any one of claims 16 or 17, wherein the optical encoding unit is adapted to optically encode a sample coordinate system, preferably an orthogonal Cartesian coordinate system, on the biological sample, and the combination of active fluorophore concentrations for the biological component is a set of coordinates in said sample coordinate system.

19. The system according to claim 18, wherein the mapping unit (90) is adapted to determine, for each separated biological component, a set of coordinates in the sample coordinate system to obtain the spatial position of each biological component in the unseparated sample (10).

Images