Method and system for determining the spatial location of a biological component in a sample

Abstract

A method for determining the spatial location of one or more biological components (12) in a biological sample (10) is proposed. The method includes the steps of optically encoding (S100) the biological components (12) in the biological sample (10) with a plurality of fluorophores (31, 32, 33, 34) to produce a combination of active fluorophore concentrations indicative of the spatial location of the one or more biological components (12), dissociating (S200) the biological components (12) from the biological sample (10) to produce dissociated biological components (12), and measuring (S310) the fluorescence of the dissociated biological components (12), mapping (S320) the fluorescence of the dissociated biological components (12) to the spatial location of the biological components (12) in the undissociated sample (10), and optically decoding (S300).

Description

[Technical Field]

[0001] Reference to other applications This application claims priority to European Patent Application EP22383262.7, filed December 22, 2022.

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

[0003] In the fields of tissue biology and cancer, it is desirable to know information about the spatiotemporal distribution of biological components, genomic states, metabolites, and gene activity in cellular machinery to understand tissue biology and cancer. To that end, a variety of different spatial omics techniques have been invented that attempt to measure genomic sequences, protein levels, epigenetic marker levels, metabolite levels, and gene expression levels in different regions of biological samples.

[0004] It is known to use markers with known spatial distribution in biological samples. In the context of spatial transcriptomics, these markers are gene product levels that can be read out using, for example, in situ hybridization or hybridization chain reaction (HCR). Each region of a sample has a unique signature of marker gene levels, and if enough marker genes are used, the region can be clearly defined. The biological sample is dissociated and processed using standard single-cell approaches, and the measured levels of these marker genes can then be used to determine the location of cells within the biological tissue.

[0005] Such methods require a priori information about how those marker genes are distributed in biological tissue. However, for mutants (including cancer samples), such a priori information is not available. Therefore, methods have been developed that do not require a priori information about the distribution of marker genes in the biological tissue of interest.

[0006] Methods that do not require a priori information have focused on creating 2D sections of biological tissue and placing the sections on slides. To perform spatial transcriptomics, for example, slides are designed with oligonucleotide signatures at known locations, and the first step of RNA-Seq library preparation is performed on the slides before tissue digestion and pooling. The signatures can then be used to determine where each read in the resulting RNA-Seq data originated within the biological sample. Alternatively, slides can be sequentially stained with probes for specific combinations of gene products and imaged to determine the spatial levels of those gene products.

[0007] However, all of these techniques are essentially two-dimensional (2D) and suffer from low capture depth (i.e., number of unique mRNAs read per cell), among other drawbacks.

[0008] For example, a publication titled "Unraveling the Complexity of the Cancer Microenvironment With Multidimensional Genomic and Cytometric Technologies" (De Vries Natasja L et al., FRONTIERS IN ONCOLOGY 2020, Vol. 10, 2020, p. 1254) teaches sectioning biological tissues and labeling patterns inherent in undisturbed tissue. Single-cell data can be obtained by flow cytometry and mass cytometry, which utilize antibodies conjugated to fluorescent dyes or heavy metal isotopes, respectively, for immunodetection of dissociated cells. For single-cell RNA sequencing, antibodies conjugated to oligonucleotides can be used to simultaneously obtain information about protein and RNA expression in single cells, which can then be mapped in space by integrating this data with spatial omics information.

[0009] The publication titled "Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics" (Longo Sophia K et al., Nature Reviews Genetics, Vol. 22, No. 10, June 18, 2021) describes the Niche-seq technology.

[0010] The two publications above propose obtaining data from two different techniques: 1) a spatial omics approach and 2) a single-cell omics approach. This means that different cells are sampled with each technique. Deconvolution or mapping from the cells used in the single-cell omics approach to the cells used in the spatial omics approach is required. This approach introduces errors because the mapping is not one-to-one and there is no guarantee that the same cell type was sampled by both techniques. Summary of the Invention

[0011] An object of the present disclosure is to provide a method for obtaining spatial information of biological components in a biological sample without a priori information.

[0012] To this end, the 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 biocomponents in a sample includes optically encoding the biocomponents in the sample with a plurality of fluorophores to create a combination of active fluorophore concentrations indicative of the spatial location of the one or more biocomponents; dissociating the biocomponents from the sample to create dissociated biocomponents of the biocomponents; and mapping the fluorescence measured from the dissociated biocomponents to the spatial location of the biocomponents in the undissociated sample.

[0014] Therefore, the present invention proposes a novel approach for determining the spatial location of one or more biological components, such as cells, in a sample. The present invention proposes optically encoding different components of a sample, such as cells, with unique signatures. By measuring and analyzing fluorescence after dissociation, it is possible to obtain the localization of the optically encoded components before dissociation of the sample.

[0015] Optically encoding biological components in a sample with multiple fluorophores to create a combination of active fluorophore concentrations that indicates the spatial location of one or more biological components imposes a coordinate system on the sample. The active fluorophore concentrations represent spatial coordinates in the coordinate system. Thus, each component of the sample has a unique sample coordinate or spatial data in the system, represented by a combination of active fluorophore concentrations.

[0016] Therefore, the combination of active fluorophore concentrations for a biocomponent is a unique signature, and this unique signature identifies the location of the biocomponent in the sample. In other words, the set of active fluorophore concentrations is a direct proxy for the spatial location of the biocomponent. Therefore, single-cell omics data and spatial data are generated from the exact same biocomponent.

[0017] On the other hand, the present invention makes it possible to obtain omics data of biological components whose spatial locations in a sample are explicitly known.

[0018] This is advantageous over prior art methods in which data is integrated from two different techniques, namely, spatial omics and single-cell omics. In prior art methods, different cells are sampled for each technique because once cells are used for one technique, they cannot be used for the other. Therefore, prior art methods require deconvolution or mapping from the cells used in the cell omics technique to the cells used in the spatial omics technique. In the present invention, both spatial and omics information for a biological component are derived from the same biological component. There is no need for any form of mapping from the spatial omics approach to the single-cell omics approach, thereby preventing the introduction of errors. Additionally, less tissue is required.

[0019] The encoded coordinate system is preferably a rectangular Cartesian coordinate system.

[0020] In one embodiment, the biological sample is chemically treated to make it optically transparent.

[0021] Optical encoding of a biological component may include staining a sample with the plurality of fluorophores and, after staining, varying the active fluorophore concentrations of some of the plurality of fluorophores in the sample. By varying the active fluorophore concentrations, a unique signature for each component can be created. A coordinate system is established for the sample. For each biological component, the unique signature is a set of sample coordinates in the coordinate system and is therefore spatial data that identifies the location of the biological component in the sample.

[0022] In one embodiment, the method comprises creating a gradient of active fluorophore concentrations in the sample, particularly where the gradients of active fluorophore concentrations are orthogonal to one another. Having three orthogonal gradients allows for marking three spatial directions in a Cartesian coordinate system.

[0023] The alteration of the active fluorophore concentration may be achieved by one of photobleaching, photoactivation, or photoswitching. A gradient can be generated that is constant across the sample.

[0024] The alterations can be performed by photobleaching, photoactivation, or photoswitching with the light sheet by exposing different parts of the sample to the light sheet for different exposure times.

[0025] In another embodiment, prior to creating multiple gradients, the ratio of fluorophore concentrations throughout the biological object is constant from component to component.

[0026] In one embodiment of the present invention, it is envisioned that at least one, and preferably two, of the plurality of fluorophores are covalently attached to the oligonucleotide.

[0027] Optical encoding can be achieved by one or more different fluorophores, where the different fluorophores have different excitation, activation, or switching spectra, and the different emission spectra define independent fluorescence channels.

[0028] An additional fluorophore can be used as a control fluorophore, provided that the control fluorophore remains unchanged while other fluorophores are altered to optically encode biological components in the sample. A step can be provided in which the measured fluorescence level of the altered fluorophore is normalized to the fluorescence level of the control fluorophore. The use of a control fluorophore can help improve the accuracy of the determination, especially when the initial staining before the fluorophore alteration is not uniform throughout the sample.

[0029] The method may include utilizing a calibration sample, where the change in concentration of each active fluorophore is recorded by imaging the calibration sample as each fluorophore is modified with increasing exposure in different tissue slices. From the resulting modification calibration graph, modification parameters for modifying the sample can be determined to determine the exposure at each axial position to achieve a desired gradient in the sample.

[0030] The method may include mapping the measured fluorescence levels of different fluorophores of the dissociated biological component to physical space in the sample before dissociation, such that each color combination measured after dissociation is assigned to a probable location in physical space.

[0031] In one aspect, mapping the measured fluorescence levels of the dissociated biological components to physical space comprises imaging the biological sample prior to dissociation and mapping the measured color space of the dissociated biological components to the measured color space in the system used to image the biological sample prior to dissociation via a histogram matching algorithm for each fluorescence channel.

[0032] Preferably, control samples stained with a single fluorophore are used to determine crosstalk between pair-wise combinations of channels in both the system used to image the sample before dissociation and the system used to measure the biocomponent signal after dissociation, and the channel data in both systems are corrected before histogram matching each channel between the two instruments.

[0033] The sample can be reconstructed from the measured fluorescence.

[0034] The present invention also provides a system for determining the spatial location of one or more biocomponents in a sample, comprising: an optical encoding unit for optically encoding biocomponents in the sample with a plurality of fluorophores to produce a combination of activated fluorophore concentrations indicative of the spatial location of the one or more biocomponents; an optical measurement system for measuring fluorescence after dissociation, the optical measurement system comprising one of a fluorescence-activated cell sorter or analyzer, a microfluidic device; and a mapping unit configured to map the measured fluorescence from the dissociated biocomponents to the spatial location of the biocomponents in the undissociated sample.

[0035] Optical encoding can be achieved by staining and modifying biological components in a sample with multiple fluorophores to create combinations of active fluorophore concentrations that indicate the spatial location of one or more biological components.

[0036] The optical encoding unit can include an imaging unit for modifying the spatial distribution of the concentrations of the plurality of fluorophores and measuring the modified spatial distribution of the concentrations of the plurality of fluorophores, and in particular, the imaging unit includes a selective plane illumination microscope for imaging the sample before dissociation.

[0037] Therefore, this document describes a method with potentially equal resolution in all dimensions. Attempting 3D omics methods with a slide-based approach poses various challenges, including alignment of successive slides, loss of spatial resolution and information at the cut surface, unequal resolution along different axes, labor time, and cost.

[0038] The method described here does not require combining spatial omics data with scRNASeq data, therefore reducing the risk of errors introduced by inaccurate mapping from single-cell RNASeq to spatial information.

[0039] This method potentially allows for greater capture depth than slide-based approaches. In fact, the method and system can be used as a module in a method pipeline. The method can be performed directly upstream of single-cell RNASeq, which means that the capture depth approaches that of scRNASeq. This is a much higher capture depth than prior art slide-based spatial omics approaches, which require supplementing spatial information from spatial omics approaches with single-cell RNA sequencing data to achieve comparable capture depth.

[0040] Finally, this method allows for the spatial information of spatial omics approaches to be combined with the capture depth of single-cell RNASeq in a single technique, thus requiring less tissue. This is particularly important in tissue-scarce situations (e.g., tumor biopsies). [Brief explanation of the drawings]

[0041] [Figure 1] 1 is a diagram showing an overview of a system according to an aspect of the present invention; [Figure 2] FIG. 1 shows an overview of a method according to one aspect of the present invention. [Figure 3] FIG. 1 illustrates a method for optically encoding according to an aspect of the present invention. [Figure 4] FIG. 1 illustrates a method for optical decoding according to an aspect of the present invention. [Figure 5] FIG. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention will now be described based on the drawings. It should be understood that the embodiments and aspects of the present invention described herein are merely examples and do not limit the scope of protection of the claims in any way. The present invention is defined by the claims and their equivalents. It will be understood that features of one aspect or embodiment of the present invention may be combined with features of different aspects or multiple aspects and / or embodiments of the present invention.

[0043] FIG. 1 shows an overview of a system 1 for determining the spatial location of multiple biological components in a biological sample 10, and FIG. 2 shows an overview of a method for determining the spatial location of biological components in a biological sample 10.

[0044] The biological sample 10 contains a biological component 12, and it is desirable to know information about the spatial distribution of said biological component 12 in the sample 10.

[0045] In the present disclosure, an example of the biological component 12 is a cell of the biological sample 10 .

[0046] The method includes a step S100 of optically encoding biological components 12 in the biological sample 10 to create a unique optical signature for each biological component 12 in the biological sample 10. In one embodiment, the biological components 12 are optically encoded in the biological sample 10 with multiple fluorophores 31, 32, 33 by creating a combination of active fluorophore concentrations that is indicative of the spatial location of the biological component in the biological sample 10. A unique signature indicative of spatial location is obtained by combining different active fluorophores, and this unique signature functions as an optical signature.

[0047] 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, this set of sample coordinates being an optical signature.

[0048] Optical encoding is described in more detail below with reference to FIG.

[0049] The method for determining the spatial location of the biological component 12 in the biological sample 10 after optical encoding includes a step S200 of dissociating the biological component 12 from the biological sample 10 to produce a dissociated biological component 12 of the biological component 12.

[0050] After dissociation, the measured fluorescence from the dissociated biocomponent 12 is mapped to the spatial location of the biocomponent 12 in the undissociated sample in step S320 to obtain the original location of the biocomponent in the undissociated sample 10. The mapping is described below with reference to FIG.

[0051] Measuring the fluorescence from the fluorophores of the dissociated biological component 12, here dissociated cells, is equivalent to reading the unique signature of the biological component. This measurement therefore makes it possible to obtain information about the spatial location of the cells before dissociation. The method then includes mapping the optical signature measured on the dissociated biological component 12 to the spatial location of cells in the undissociated sample 10.

[0052] The set of active fluorophore concentrations is a direct proxy for the spatial location of the biocomponent in the biological sample, thus generating single-cell omics data and spatial data from the very same biocomponent.

[0053] Those skilled in the art will appreciate that the present invention does not suffer from the drawbacks of known 3D imaging methods using slide-based approaches, such as the challenges of registering successive slides, loss of spatial resolution and information at cross sections, uneven resolution along different axes, labor time, and cost.

[0054] Optical encoding will now be described in detail with reference to FIG.

[0055] First, the biological sample 10 is prepared. This preparation includes, in step S110, fixing and permeabilizing cells (as an example of biological components) of the biological sample 10 using a fixative and a permeabilizing agent.

[0056] Suitable fixatives include, but are not limited to, paraformaldehyde / formalin, methanol, acetone, DSP and its derivatives, and quick freezing.

[0057] The sample 10 can be permeabilized with a permeabilizing agent. Suitable permeabilizing agents include, but are not limited to, organic solvents and detergents in general, such as Triton X-100, Tween-12, CHAPS, Sarkosyl, DMSO, SDS, and saponin. Some of these agents can simultaneously fix and permeabilize the sample 10.

[0058] Next, in step S120, the biological sample 10 is stained with a plurality of fluorophores 31, 32, 33, 34. The staining is carried out with at least three different fluorophores 31, 32, 33. The three different fluorophores 31, 32, 33 have different excitation spectra and different emission spectra.

[0059] In a detailed example, three different fluorophores are used to create a three-dimensional coordinate system, however this is not a limitation of the invention and other coordinate systems could be envisaged.

[0060] 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.

[0061] The fourth fluorophore 34 is also used as a control fluorophore. The concentrations of the four different fluorophores are stoichiometric, i.e., the concentrations of the fluorophores are proportional to one another. Therefore, the fourth (control) fluorophore 34 allows normalization of the encoding fluorophore concentrations 31, 32, and 33. The use of a control fluorophore or control channel relaxes the requirement for perfectly spatially uniform fluorophore concentrations. Thus, the fourth fluorophore 34 is a staining control used to normalize the signals from the other three fluorophores, allowing for control of different staining efficiencies in different parts of the sample 10.

[0062] The initial fluorophore concentrations 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.

[0063] Suitable fluorophores include, for example, cell marker fluorophores that bind to genomic DNA or RNA, cell membranes, and any other components of the cell, and that are covalently attached directly to oligonucleotides or indirectly attached through a process involving a probe, including, but not limited to, padlock probes, snail probes, nanoballs generated by hybridization chain reaction, or rolling circle amplification.

[0064] When the stains are oligonucleotides or probes that bind directly or indirectly to genomic DNA, these fluorophores (31, 32, 33, 34) can be covalently attached to oligonucleotides whose sequences are found multiple times in the genomes of multiple species.

[0065] When the stains are oligonucleotides or probes that bind directly or indirectly to RNA, these fluorophores (31, 32, 33, 34) can be covalently attached to oligonucleotides or probes whose sequences occur multiple times in the expressed RNA.

[0066] A suitable hybridization mixture for staining biological subject 10 includes, but is not limited to, components such as DMSO, Triton X-100, Tween-12, sodium citrate saline, dextran sulfate, heparin, SDS, formamide, Denhardt's solution, and EDTA.

[0067] If the staining agent is an oligonucleotide, the biological sample 10 may be heated and cooled one or more times to promote annealing.

[0068] In one embodiment, it is contemplated to have two oligonucleotides of identical sequence, each with two fluorophores covalently attached.

[0069] The fluorophores 31, 32, 33, 34 are positioned as far apart as possible on the oligonucleotide and / or as spectrally distinct as practical to avoid non-radiative energy transfer.

[0070] It should be noted that when fluorophores 31, 32, 33, and 34 are covalently attached directly or indirectly to oligonucleotides, one or more of fluorophores 31, 32, 33, or 34 can be attached to any given oligonucleotide. When fluorophores 31, 32, 33, and 34 are attached to the same oligonucleotide, optimizing the technique requires maximizing the physical and spectral distance between fluorophores 31, 32, 33, and 34. When fluorophores 31, 32, 33, and 34 are attached directly or indirectly to oligonucleotides, the sequences of those oligonucleotides should be found in the genome of the species of biological subject 10 to be stained.

[0071] In the example of oligonucleotides covalently linked to fluorophores or probes, they can be annealed to the genomic DNA or RNA of the biological components 12 of the biological sample 10 by heating and cooling steps.

[0072] It should be noted that after staining, the biological sample 10 can be washed to remove unbound staining agent and post-fix before changing the fluorophore concentration. Suitable washes 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 in further steps of the process.

[0073] The next step is to prepare the biological sample 10 for the optical encoding process. This can include mounting the sample 10, possibly dehydrating it for the 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 the BABB and DISCO families, require the sample 10 to be dehydrated before being cleared by immersion in an organic solvent.

[0074] Suitable mounting agents include, but are not limited to, low melting point agarose, agarose, polyacrylamide, and Matrigel.

[0075] Suitable dehydrating agents include, but are not limited to, methanol, ethanol, propanol, and tetrahydrofuran (THF).

[0076] Suitable clearing agents and methods include, but are not limited to, BABB, CLARITY, PACT, PARS, 3DISCO, iDISCO, Spalteholz, glycerol, and CUBIC. For simplicity, the remainder of this detailed description of the invention will describe clearing as being performed using the BABB protocol, although other clearing protocols may also be used.

[0077] The next step in the process, step S130, is to image and vary the active concentrations of fluorophores 31, 32, 33, 34, thereby creating a gradient of active fluorophore concentrations within biological sample 10. This step S130 can be performed using optical encoding system 50, which is used to vary and / or image the distribution of active fluorophores.

[0078] There are multiple mechanisms that can be used to optically alter the active fluorophore concentration. Depending on the choice of fluorophore used, these mechanisms include, but are not limited to, photobleaching, photoswitching, or photoactivation. For brevity, in this detailed description of the invention, we will describe the use of photobleaching.

[0079] One way to perform optical encoding S100 is to use an optical instrument that utilizes spatially non-uniform illumination scanning. The desired active fluorophore distribution in the sample 10 results from the particular non-uniform illumination and scanning pattern utilized. Examples of such instruments include, but are not limited to, a light sheet microscope, a confocal microscope, or a two-photon microscope. For simplicity, in the remainder of this detailed description of the present invention, we will describe the imaging and modification (i.e., optical encoding) in step S130 as being performed using a SPIM microscope.

[0080] An alternative to using scanning instruments for optical encoding is to use 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 the need for scanning.

[0081] The multiple gradients are created after the staining step S120 by a step S130 of varying the active fluorophore concentrations of some of the multiple fluorophores 31, 32, 33, 34 in the biological sample 10.

[0082] Optimally, the gradients of active fluorophore concentrations are orthogonal to one another: a first gradient for a first fluorophore having a first excitation spectrum and a first emission spectrum, a second gradient for a second fluorophore having a second excitation spectrum and a second emission spectrum, and a third gradient for a third fluorophore having a third excitation spectrum and a third emission spectrum.

[0083] In other words, each cell can be identified by its unique optical signature represented by three fluorophores (one fluorophore per dimension).

[0084] It will be appreciated that a minimum of three fluorophores is required to unambiguously encode each of the three spatial dimensions. An optical signature is a set of sample coordinates represented by three fluorophores (one fluorophore per dimension). Coordinate systems with additional spatial dimensions are also contemplated.

[0085] A calibration sample is first imaged in 3D in all four channels to confirm successful staining.

[0086] The concentration of the active fluorophores 31, 32, 33, 34 can then be altered via photoactivation, photoswitching, or photobleaching, depending on the type of fluorophore 31, 32, 33, 34 used. The resulting concentration distribution of the active fluorophores 31, 32, 33, 34 may include one that provides distinct location information for one or more cells within the biological sample 10.

[0087] In one embodiment, a selective plane illumination microscope (SPIM) or other light sheet microscope 50 is used to image the cells. The calibration sample is bleached in a single plane, i.e., for each channel or for each channel to be bleached, in a different, non-overlapping plane in a separate portion of the calibration sample.

[0088] The decrease in concentration of active fluorophores 31, 32, 33, and 34 is recorded by imaging the biological sample 10 as the sample bleaches over time. A bleach calibration graph 48 is generated that determines the bleaching parameters required for each of the fluorophores 31, 32, 33, and 34 to bleach to a desired level of active fluorophore concentration. The bleaching parameters for each of the three channels are read from the calibration graph generated by bleaching each of the fluorophores 31, 32, 33, and 34 in a calibration sample. The bleach calibration graph 48 can be used to determine the exposure at each axial position to achieve the desired gradient in the biological sample 10.

[0089] Light sheet microscope 50 rapidly bleaches a predetermined plane within biological sample 10 while leaving the remainder relatively unaffected. When light sheet microscopes are used, the light sheet can be generated, for example, via a cylindrical lens or by a fast-moving laser line via a galvanometer mirror or resonant scanner. In addition, light sheet microscopes can easily generate 3D images of the resulting bleaching pattern, allowing the resulting active fluorophore distribution to be confirmed.

[0090] Post-bleach imaging of the biological sample 10 can be used to map the fluorescence measured from the dissociated biological components 12 to the spatial location of the biological components 12 in the undissociated sample, as described later in this disclosure. Post-bleach imaging of the biological sample 10 can be used for mapping, along with a single color control to determine the color space mapping between the imaging / modification system and the dissociated cell detection.

[0091] As mentioned above, it is desirable to create three orthogonal gradients, i.e., orthogonal monotonic distributions, for example. In practice, linear distributions tend to optimize signal-to-noise ratios and spatially uniform positional precision. After writing the desired pattern into the fluorophores, the pattern is imaged in 3D and the actual pattern obtained is quantitatively recorded.

[0092] Before altering the intensities of the fluorophores, the Pearson correlation coefficient should be strong for all of the altered fluorophores 31, 32, 33 relative to the control fluorophore 34 used, 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.

[0093] One technique for performing step S130 of varying the active fluorophore concentration is photobleaching with a light sheet 40 by exposing different portions of the biological sample (10) to the light sheet (40) for different exposure times.

[0094] The bleaching parameters for each of the three channels are derived from a calibration graph generated from bleaching each fluorophore in a calibration sample.

[0095] One approach consists of generating a linear distribution of active fluorophores that results in uniform resolution across the entire axis. Because the generated gradient is linear rather than exponentially decaying, the position error will be uniform at any position along the axis. If a gradient with exponential decay were generated, for example, by varying the exposure linearly along the axis, the amount of position error along the axis would vary depending on the slope of the curve.

[0096] The first dimension x is bleached using a light sheet perpendicular to the x dimension that excites one of the fluorophores 31 that resides for different times within the biological sample 10 .

[0097] The longer the light sheet is placed at each position, the lower the concentration of unbleached fluorophores in the corresponding plane. Alternatively, different regions of the biological sample 10 can be scanned different times to create bleaching gradients, or the laser power can be spatially varied. Multiple scans are essentially the same as varying the exposure time.

[0098] The second dimension, y, is bleached by rotating the sample 90 degrees about the vertical axis (z) and then repeating the process used to bleach the x dimension using a laser line appropriate for the second fluorophore 32.

[0099] The third dimension, z, is bleached by scanning a finite height light sheet with a laser line appropriate for the third fluorophore 33 over the biological sample 10 for equal periods of time. Between scans, the sample 10 is gradually raised (translated along the z axis) out of the path of the light sheet, so that different regions within the sample are illuminated at different times and a gradient of the active fluorophore 33 is bleached along the third dimension.

[0100] 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 used to bleach the y dimension can be repeated using a laser line appropriate for the third fluorophore 33.

[0101] An alternative to the steps 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, is to provide an instrument capable of illumination along more than one axis, thereby allowing sequential or simultaneous bleaching along multiple axes without the need to rotate the sample between bleachings.

[0102] The result is a biological sample 10 with orthogonal active fluorophore gradients along three spatial dimensions and an unbleached control fluorophore 34 .

[0103] After bleaching, the concentration profile of the four fluorophores is determined by imaging the biological sample 10 in three dimensions in all four channels.

[0104] This is not the only way, and the alteration can be done by one of photoactivation or photoswitching.

[0105] The next step is to "unclear" the sample, returning it to its pre-cleared state. This can be done by reversing the clearing protocol, or, for protocols that required dehydration, by rehydration as needed. Suitable unclearing 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.

[0106] The next step involves dissociating the biocomponents 12 from the biological sample 10 in step S200 to create individual biocomponents 12 of the biocomponents 12.

[0107] The sample 10 may be enzymatically dissociated using enzymes including, but not limited to, collagenase, dispase, and trypsin. Tissue may also be mechanically dissociated by vortexing, sonication, manual or electric pestles, or the use of specialized tissue dissociators such as the gentleMACS. A combination of enzymatic and mechanical methods may also be utilized to dissociate the biological sample 10.

[0108] The next step involves separating the biocomponents 12 into separate compartments for downstream processing. Biocomponents can be separated using a FACS machine, a microfluidic device, or by dilution. Compartments include wells in multiwell plates, fabricated microwells, tubes, spots on slides, and bubbles in water-in-oil emulsions.

[0109] After dissociation, the next step is optical decoding S300, which includes measuring the fluorescence of the dissociated biocomponents S310 and mapping those fluorescence measurements to the spatial location of the biocomponents in the undissociated sample S320.

[0110] This is now explained below with reference to FIG.

[0111] First, the fluorescence from the dissociated biological component 12 is read. More precisely, during or after separation of the components, the fluorescence intensity of the multiple fluorophores should be read to determine where in the sample 10 the biological component 12 originated.

[0112] To read the intensities of the fluorophores 31, 32, 33, 34, a measurement system (80) can be used that includes an excitation system 60, such as a laser, LED, or fluorescent lamp, to excite the fluorophores, and a detector system 70, such as a camera, PMT, or other type of photodetector, of an optical decoding system 300, to measure the fluorescence. This detection (step S310) can be performed, for example, in a fluorescence-activated cell sorting (FACS) machine, a fluorescent plate or slide reader, or a fluorescence excitation / detection setup integrated within a microfluidic device.

[0113] Any omics can then be performed on the biological components 12 within the individual compartments, with the location of each component 12 within the original biological sample 10 being derived from the three-axis fluorescence levels normalized to the control channel.

[0114] The measured fluorescence levels are mapped in step S320 to the spatial location of undissociated cells in the biological sample 10. Each color combination measured after dissociation is thereby assigned to a unique location in physical space within the biological sample 10. In effect, the set of active fluorophore concentrations is a direct proxy for the spatial location of the biological component in the biological sample.

[0115] For each biological component 12, the levels recorded in the three bleached channels F1, F2, and F3 can be normalized by the level of the control, non-bleached channel F4. Normalization means dividing the levels of the bleached channels F1, F2, and F3 by the level of the control channel F4.

[0116] Typically, a linear distribution of active fluorophores can be used, and the resulting optical signature is a direct proxy for the original location of the biological component 12 within the biological sample 10. The mapping between the normalized "color code" of the cells (F1 / F4, F2 / F4, F3 / F4) and the original x, y, z location of the cells within the biological sample 10 is a simple set of linear equations.

[0117] In other words, the original 3D location of the biological components 12 is reconstructed by normalizing the level of active fluorophores in each altered bleaching, photoactivation, or switching channel to that of a control, unaltered channel to produce an optical signature for each component 12. The resulting optical signature serves as a proxy for the location in physical space, i.e., the original location {x,y,z} of each component 12 within the biological sample 10. This approach can be performed if the distribution of active fluorophore concentrations is an underlying optically encoded form in linear algebra terms.

[0118] The optical encoding imparts a coordinate system to the biological sample, and the concentration of the active fluorophore for a biological component becomes a set of spatial coordinates in the coordinate system.

[0119] Prior to dissociation, it is possible to measure the color-coded distributions or values ​​(I1, I2, I3, and I4) with the light sheet microscope 50 used for bleaching. As noted above, for orthogonal linear distributions of active fluorophores, this mapping from "color space" to "physical space" is linear.

[0120] After dissociation, the fluorescence levels (F1, F2, F3, and F4) in the dissociated cells can be measured and recorded, for example, by a detector system 70, for example, a FACS machine.

[0121] Ideally, the response of the system (e.g., light sheet microscope 50) used to image the biological sample 10 before dissociation but after altering the distribution of the active fluorophores, and the response of the detector system 70 (e.g., FACS machine) used to detect fluorescence levels in the dissociated cells, would be identical for a given active fluorophore concentration.

[0122] Those skilled in the art will understand that even if one attempts to match as closely as possible the responses measured in the optical encoding system used to alter and / or image the distribution of active fluorophores with the responses measured in the system used to read out the fluorescence after dissociation of biological component 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 out the fluorescence after dissociation of biological component 12 is a detector system 70, similar laser lines can be used for excitation in light sheet microscope 50 and detector system 70 to match the responses as closely as possible. However, the responses will not be identical. This may be due to, for example, slightly different lasers, completely different detectors, different filters, and different sample media.

[0123] It is therefore proposed to have a mapping from the color space of the fluorescence recorded after dissociation {F1 / F4, F2 / F4, F3 / F4} to the color space of the fluorescence recorded before dissociation {I1 / I4, I2 / I4, I3 / I4}, which can then be mapped to the physical space {x,y,z} as above.

[0124] Therefore, mapping the measured color space of dissociated cells to physical space, i.e., mapping {F1,F2,F3,F4} → {x,y,z}, involves first mapping the measured color space of dissociated cells to the imaged color space in the imaging system 50 used to image the sample 10, i.e., mapping {F1,F2,F3,F4} → {I1,I2,I3,I4}. Mapping the imaged color space to physical space, {I1 / I4,I2 / I4,I3 / I4} → {x,y,z}, is straightforward because we have a voxel dataset from the post-bleach sample scan: in this voxel data, voxel coordinates represent the physical space {x,y,z} and voxel values ​​represent the imaged color space {I1 / I4,I2 / I4,I3 / I4}. This mapping from the measured color space of dissociated cells to physical space is performed by using four calibration samples, each containing only one of the four fluorophores 31, 32, 33, and 34, to calibrate the response of the system 50 used to modify and / or image the distribution of the active fluorophores and the system 80 used to read out the fluorescence after the biocomponent 12 has dissociated. The mapping between the response curves of the imaging system 50 and the detector system 80 then gives the desired relationship between the color space of the fluorescence recorded after dissociation {F1,F2,F3,F4} and the color space of the fluorescence recorded before dissociation {I1,I2,I3,I4}, and therefore the desired relationship to 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, a histogram matching algorithm for each channel, or the use of artificial intelligence methods.

[0125] Histogram matching is performed using the fluorescence signals measured in all four channels of four biological samples, each stained with only a single type of fluorophore, for both the system used to alter and / or image the distribution of active fluorophores and the system used to read out the fluorescence after dissociation of the biological components 12. The measured optical signature of each biological component 12 after dissociation is then mapped to the corresponding optical signature in the system used to image the 3D fluorophore distribution before dissociation.

[0126] The resulting fitted optical signature can be compared to the fluorophore concentration at every physical location in the 3D image (after altering the distribution of active fluorophores) using a distance metric such as Euclidean distance, and the pre-dissociation physical location of the biocomponent 12 can be retrieved from the optical signature by selecting the most likely location where this metric is minimal. Other suitable distance metrics for this mapping include, but are not limited to, Pearson, Kendall's tau, Manhattan, and Spearman's rank correlation distance.

[0127] Although optical signatures may not be encoded as monotonic gradients, they may be used in some other way to provide unambiguous location information. For example, if four or more suitable fluorophores are available, combining two fluorophores along an axis may provide unambiguous location even if one fluorophore alone does not provide unambiguous information. In this case, a mapping step may instead be used in which the ensemble of normalized optical signatures of biocomponents 12 is compared to the optical signatures encoded in the sample 10 (which are read out during the imaging stage after bleaching) using any form of appropriate distance metric, such as Euclidean distance, to map the resulting biocomponents 12 to their most likely location of origin in the sample 10. Other suitable distance metrics for this mapping include, but are not limited to, Pearson's, Kendall's tau, Manhattan, and Spearman's rank correlation distance.

[0128] Once enough biological components 12 have been mapped to their expected locations of origin within the biological sample 10, the original tissue can be reconstructed (S400).

[0129] FIG. 5 shows an example of data obtained using the method and system of the present disclosure using the protocol detailed below.

[0130] Imaging and bleaching Samples were imaged and bleached using a homemade cylindrical lens-based OPTiSPIM light sheet mesoscope. Briefly, 50 mW 405 nm, 50 mW 488 nm, and 50 mW 639 nm lasers were used to image and bleach Alexa405, FAM, and Alexa647 fluorophores, respectively. A 5 mW 543 nm laser was used to image the internal staining control, TAMRA fluorophore. Bandpass filters of 447BP60, 525BP50, 585BP60, and 700BP75 were used to detect Alexa405, FAM, TAMRA, and Alexa647 fluorophores, respectively. A 2.5x N Plan-air objective (Leica, NPLAN, NA = 0.07) was used to illuminate the sample. For detection, a 5xN Plan 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.

[0131] The light sheet for each laser line was placed at a fixed position and bleaching was calibrated using calibration samples by monitoring the decrease in the intensity of the fluorescent signal with exposure time. The bleaching exposure times at different positions of the light sheet in the test sample were determined by reading from these calibration graphs (one for each fluorophore / laser line).

[0132] Treatment of faded 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 with one quick wash and two longer washes (>4 hours) in PBS. For experiments analyzing limb ectoderm and mesenchyme separately, limb buds were incubated in 0.5% trypsin-EDTA 10x (phenol red-free - Gibco 15400-054) for 15 minutes at room temperature, after which the ectoderm was removed from the limb with tweezers. For all other experiments, tissues were digested with 0.22 μm-filtered 10 mg / ml collagenase / dispase in PBS (containing 1 / 1,000,000 Triton) at 37°C and 600 RPM for 2 hours. Biological samples 10 were periodically mechanically agitated with a Gilson pipette. After tissue dissociation, sample 10 was centrifuged at 600 g for 5 minutes at room temperature. The cell pellet was resuspended in 0.5-1 ml of PBS, filtered through a 40 μm strainer, and processed on a FACS analyzer (Fortessa). Note that this is not a limiting example and other devices, such as a cell sorter (FACSAria), can also be used.

[0133] Post-dissociation detector system In the example shown, cells were simply analyzed for their fluorescence level using a Fortessa FACS analyzer. To remove debris, a FACS gate on side scatter area and forward scatter was used (gate P1). Singlets were then selected with a forward scatter height vs. area gate (gate P2). A PE-A (corresponding to a 586BP15 bandpass filter to detect the TAMRA staining control) vs. forward scatter gate was used to select fluorescent cells and remove remaining debris (gate P3).

[0134] The fluorescence channel levels of PACB (corresponding to a 450BP50 bandpass filter for detecting Alexa405), FITC-A (corresponding to a 530BP28 bandpass filter for detecting FAM), and APC-A (corresponding to a 670BP14 bandpass filter for detecting Alexa647) were normalized to the PE-A (to detect TAMRA) fluorescence channel level to generate the data shown in Figure 5. Therefore, for simplicity, in this example, the reconstructions assumed identical responses between the FACS and SPIM instruments, and linear gradients were generated in all dimensions, so that color space is a direct proxy for physical space (as explained in paragraphs 104-107).

[0135] Figure 5 shows the results of reconstructing an E10.5 hind limb bud from a mouse embryo that underwent the C3PO process in all three dimensions. Before dissociation, ectodermal cells are known to form a cap over the mesenchyme, with a central depression and a thickened, curved edge corresponding to the well-characterized apical ectodermal ridge (AER). The top left panel of Figure 5 shows that ectodermal cells are indeed found in a curved thickening around the exact location where the AER is expected (low x-dimension). The top right panel shows a transverse slab of ectodermal cells, with a clear depression in the center of the ring of ectodermal cells, as expected. Furthermore, almost no ectodermal cells are detectable along the linear edge of the limb bud (high x-dimension). This is where the limb bud detaches from the rest of the embryo, and no ectoderm should be present along this edge, as seen in the reconstruction. Finally, the bottom panel shows limb ectodermal cells (large dots) and limb mesenchymal cells (small dots). Because in wild-type limb buds a layer of ectodermal cells lies directly on top of the mesenchyme, the ectodermal cells should surround the mesenchyme, which is exactly the configuration observed in the reconstruction. Overall, therefore, the reconstructed configuration of the limb bud is very similar to that before dissociation, demonstrating that the method described in this disclosure is functional.

[0136] Reference Number List 1 System 10. Biological samples. Examples include, but are not limited to, organs, embryos, or biopsy tissue. 12. Biological components. Examples include, but are not limited to, cells or cell masses. 31,32,33 Fluorophores 34 control fluorophores 40 Light Sheet 48 Calibration Graph Systems used to modify and / or image the distribution of 50 active fluorophores 60 Excitation System 70 Detection System 80 Optical measurement system used to read out fluorescence after cells are dissociated 90 mapping units 100 Optical Encoding Unit 300 Optical Decoding System Color space of fluorescence recorded using (70) after dissociation of {F1,F2,F3,F4} {I1,I2,I3,I4} color space of fluorescence recorded using (50) before dissociation {x,y,z} physical space

Claims

1. 1. A method for determining the spatial location of one or more biological components (12) in a biological sample (10), comprising: - optically encoding (S100) biological components (12) in a biological sample (10) with a plurality of fluorophores (31, 32, 33, 34) to create a combination of active fluorophore concentrations within the biological sample (10) that indicates the spatial location of one or more biological components (12); - dissociating (S200) the biological component (12) from the biological sample (10) to produce a dissociated biological component (12); and - measuring the fluorescence of the dissociated biocomponent (12) (S310), mapping it to the spatial location of the biocomponent (12) in the undissociated sample (10) (S320) and optically decoding it (S300); A method comprising:

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

3. 3. The method of claim 2, wherein the optical decoding step includes determining a set of coordinates in the sample coordinate system for each dissociated biological component to obtain the spatial location of each biological component in the undissociated sample (10).

4. The method of any one of claims 1 to 3, wherein creating a spatial combination of active fluorophore concentrations comprises staining (S120) a biological sample (10) with the plurality of fluorophores (31, 32, 33, 34).

5. 5. The method according to any one of claims 1 to 4, wherein a plurality of different fluorophores are covalently attached to a single molecule, e.g., an oligonucleotide, in particular wherein the plurality of different fluorophores are attached to portions of the biological component in a fixed ratio.

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

7. The method according to any one of claims 1 to 6, comprising creating a gradient of concentrations of a plurality of active fluorophores in the biological sample (10), in particular the gradients of the concentrations of a plurality of active fluorophores being orthogonal to each other.

8. The method of claim 6 or 7, wherein changing the active fluorophore concentration (S130) is performed by one of photobleaching, photoactivation, or photoswitching, and in particular changing (S212) is performed by photobleaching using a light sheet (40) by exposing multiple areas of the biological sample (10) to the light sheet (40) for different exposure times.

9. 9. The method of claim 8, comprising measuring the fluorescence levels of calibration samples before and after varying the active fluorophore concentration to generate a bleaching calibration graph (48) that defines bleaching parameters to use when varying the active fluorophore concentration in the target sample (10).

10. 10. The method according to any one of claims 1 to 9, wherein the optically encoding step (S100) is performed by means of different fluorophores (31, 32, 33) having different emission spectra and different excitation spectra that define at least three fluorescence channels.

11. 11. The method of any one of claims 1 to 10, comprising using an additional fluorophore (34) as a control fluorophore, and in particular further comprising normalizing the measured fluorescence levels of the non-control fluorophores (31, 32, 33) to the fluorescence level of the control fluorophore (34).

12. 12. The method according to any one of claims 1 to 11, comprising mapping the measured fluorescence levels of different fluorophores (31, 32, 33) of the dissociated biological component (12) onto physical space in the biological sample (10) before dissociation, such that each color combination measured after dissociation is assigned to a probable location in physical space.

13. 12. The method of claim 11, wherein mapping the measured fluorescence levels of the dissociated biological component (12) to physical space comprises imaging the biological sample (10) before dissociation and mapping the measured color space of the dissociated biological component (12) to a measured color space in a system used to image the biological sample (10) before dissociation, e.g., by a histogram matching algorithm for each fluorescence channel.

14. 14. The method of claim 12 or 13, wherein a control sample stained with a single fluorophore is used in both the system used to image the biological sample (10) before dissociation and the system used to measure the signal of the biological component (12) after dissociation to determine crosstalk between pairwise combinations of channels and to correct the channel data in both systems before histogram matching each channel between the two instruments.

15. The method of any one of claims 1 to 14, further comprising reconstructing the biological sample (10) from the measured fluorescence.

16. 1. 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) provided for optically encoding biological components (12) in the biological sample (10) with a plurality of fluorophores (31, 32, 33, 34) to produce a combination of active fluorophore concentrations indicative of the spatial location of one or more biological components (12); - an optical measurement system (80) for measuring fluorescence after dissociation, comprising one of the following: a fluorescence activated cell sorter or analyzer, a microfluidic device; a mapping unit (90) configured to map the measured fluorescence from the dissociated biocomponent (12) to the spatial location of the biocomponent (12) in the undissociated sample (10); Including, the system.

17. The system of claim 16, wherein the optical encoding unit includes an imaging unit for modifying the spatial distribution of the concentrations of multiple fluorophores and measuring the modified spatial distribution of the concentrations of multiple fluorophores, and in particular the imaging unit includes a selective plane illumination microscope or other light sheet microscope (50) for imaging the biological sample (10) before dissociation.

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

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

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