{"id":10730,"date":"2025-04-22T10:00:18","date_gmt":"2025-04-22T10:00:18","guid":{"rendered":"https:\/\/fluorofinder.com\/?p=10730"},"modified":"2025-04-22T18:51:32","modified_gmt":"2025-04-22T18:51:32","slug":"spatial-omics","status":"publish","type":"post","link":"https:\/\/fluorofinder.com\/spatial-omics\/","title":{"rendered":"The Rise of Spatial-Omics Technologies"},"content":{"rendered":"

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Single-cell analysis techniques such as fluorescence-activated cell sorting (FACS) and single-cell DNA and RNA sequencing have broadened researchers\u2019 understanding of cell biology in health and disease by enabling the study of rare cell types and phenotype variations within same-cell populations. However, new methods are evolving rapidly, driven by the need for molecular profiling of individual cells within their native spatial context. Here, we look at some of these so-called spatial-omics technologies, including key protocol differences. We also share insights from Lindsy Rapkin, PhD, Field Applications Scientist at Standard BioTools\u2122, who explains how Imaging Mass Cytometry\u2122 (IMC\u2122) has been combined with RNAscope\u2122 in situ <\/em>hybridization (ISH) technology to simultaneously visualize key protein and RNA markers in tissue samples.<\/p>\n

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Figure 1. <\/strong>IMC staining of pancreatic ductal adenocarcinoma (PDAC) tissue showing 7 markers: aSMA (red), HLADR (yellow), E-cadherin (cyan), collagen 1 (green), CD68 (magenta), INOS (blue) and Granzyme B (white).<\/p>\n

What is Spatial-Omics?<\/strong><\/h3>\n

Omics refers to a broad range of technologies used for characterizing different biomolecules in cells or tissues. Specifically, genomics technologies allow for studying the whole genome of a cell or organism; epigenomics technologies are used for investigating chemical modifications to DNA, which can alter gene expression; and transcriptomics technologies enable the analysis of RNA transcripts. Proteomics and metabolomics technologies are respectively used for characterizing proteins and metabolites. Spatial-omics is the application of one or more of these technologies in situ<\/em>, typically using formalin-fixed, paraffin-embedded (FFPE) tissue samples. Bringing spatial context to omics data is important since the functional state of any given cell can be modulated by its neighbors, either through direct interaction, via signaling molecules or by microenvironmental factors such as chemical compound gradients.<\/p>\n

Spatial-Omics Technologies<\/strong><\/h3>\n

Established methods for spatial profiling include immunohistochemistry (IHC), which uses fluorophore-labeled antibodies for detecting proteins of interest, and fluorescence in situ <\/em>hybridization (FISH), which relies on fluorescent probes to bind specific DNA target sequences. Yet, while these techniques still have broad applicability for scientific research, they are now complemented by a vast array of more than 50 advanced technologies that have been developed to overcome known limitations of existing methods1<\/sup>. With so many different options available for spatial profiling, it is impossible to cover every technology here. As such, the following are some of the main approaches on which these newer modalities are based:<\/p>\n

Cyclical IHC with Fluorophore-Labeled Antibodies<\/strong><\/h4>\n

A major drawback of traditional immunofluorescent IHC is that it allows for detecting only a handful of protein markers due to spectral overlap. However, it has been found that the multiplexing capacity of IHC can be increased by performing sequential rounds of staining, imaging, and stripping\/bleaching. Methods based on this strategy include cyclic immunofluorescence (cycIF), which involves mild chemical inactivation of common dyes after each image acquisition; iterative bleaching extends multiplexity (IBEX), which rapidly bleaches fluorophores without causing epitope loss or tissue destruction; and MACSima\u2122 Imaging Cyclic Staining (MICS), which incorporates both photobleaching and the controlled release of antibodies (REAlease\u00ae Antibodies) or their labels (REAdye_lease\u2122 Antibodies)2,3,4<\/sup>. These techniques allow for detecting tens to hundreds of different protein targets in the same tissue sample but require careful implementation to avoid compromising antibody binding.<\/p>\n

Use of DNA-Barcoded Antibodies<\/strong><\/h4>\n

Like fluorophore-labeled antibodies, DNA-barcoded antibodies can be employed for detecting proteins of interest, typically through their use with fluorescently labeled complementary DNA probes. A technology based on this approach known as immunostaining with signal amplification by exchange reaction (Immuno-SABER) has been shown to achieve up to 180-fold signal amplification using standard detection equipment5<\/sup>. Another method termed co-detection by indexing (CODEX) involves iterative cycles of staining and imaging and is claimed to have unlimited multiplexing capacity6<\/sup>.<\/p>\n

mRNA Probes for Targeted Transcripts<\/strong><\/h4>\n

mRNA probes have been widely adopted for spatial transcriptomics, with some methods now able to detect up to 10,000 transcripts. This high multiplexing is achieved using a combination of spectral barcoding, whereby different fluorophores are paired with specific mRNA target sequences, and temporal barcoding, which is based on multiple rounds of probe hybridization and stripping1<\/sup>. Examples of spatial transcriptomics technologies developed within the last decade include multiplexed error robust fluorescence in situ<\/em> hybridization (MERFISH), sequential fluorescence in situ<\/em> hybridization (seqFISH), and Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA). These serve as popular alternatives to the established method of performing laser capture microdissection (LCM) followed by single-cell RNA sequencing (scRNA-seq), which has a complex and time-consuming workflow.<\/p>\n

Imaging Mass Cytometry<\/strong><\/h4>\n

Imaging Mass Cytometry, based on CyTOF\u00ae technology, uses antibodies labeled with metal tags for spatial proteomics. \u201cOnce the sample has been immunostained, it is introduced into a Hyperion XTi\u2122 Imaging System<\/a>, where it is ablated by a UV laser, one pixel at a time, before being transferred to an inductively coupled plasma for ionization,\u201d Rapkin explains. \u201cThe ions are then quantified and the signals corresponding to each tag are correlated back to the respective markers.\u201d A major advantage of using metal tags instead of fluorophores is that there is no need to worry about spectral overlap or autofluorescence. As a result, IMC allows for imaging as many as 40 or more markers simultaneously, making it more efficient than cyclical IHC methods.<\/p>\n

Multimodal Spatial Profiling with IMC and RNAscope<\/strong><\/h4>\n

Standard BioTools has developed a simple three-step workflow for co-detecting proteins and RNA with a combination of IMC and RNAscope ISH Technology (from Advanced Cell Diagnostics, a Bio-Techne brand). First, oligonucleotides are labeled for RNAscope. The RNAscope assay is then performed, and the samples are incubated overnight with IMC antibodies. A standard image analysis pipeline is used for deciphering the data.<\/p>\n

\"\" \"\" \"\"<\/p>\n

Figure 2.<\/strong> The integrated IMC and RNAscope workflow.<\/p>\n

\u201cWhile detecting protein targets allows for determining cellular identity, interrogating the transcriptome can provide a deeper understanding of cellular function and activation state,\u201d Rapkin says. \u201cBy applying IMC and RNAscope for spatial profiling within the tumor microenvironment, we have demonstrated the value of multimodal spatial profiling for investigating tumor-immune interactions. This strategy could readily be applied across other fields of research for deeper insights into sample material.\u201d To learn more, visit https:\/\/www.standardbio.com\/resources\/blog-articles\/2023\/06\/imc-rnascope-poster<\/a>.<\/p>\n

\"\"<\/p>\n

Figure 3. <\/strong>IMC and RNAscope co-detection of a breast cancer sample. IMC (left panel) showing the structural markers Collagen 1 (red), E-cadherin (green), and aSMA (yellow), plus DNA (blue). RNAscope (middle panel) showing UBC (red), B2M (green), and GAPDH (blue). Maxpar IMC Cell Segmentation kit staining (right panel).<\/p>\n

Technologies for Epigenomics and Metabolomics<\/strong><\/h4>\n

Although many different technologies have been developed for spatial proteomics and transcriptomics, fewer methods exist for spatial epigenomics and metabolomics. Available options for epigenomics research include spatial-ATAC-seq, which provides spatially resolved chromatin accessibility profiling via next-generation sequencing, and Spatial-CUT&Tag, which delivers genome-wide profiling of histone modifications7,8<\/sup>. For metabolomics, metaFISH pairs FISH with matrix-assisted laser desorption\/ionization imaging mass spectrometry (MALDI-IMS) to map metabolic species in native tissues, while airflow-assisted desorption electrospray ionization (AFADESI)-IMS provides untargeted analysis of more than 1,500 metabolites9,10<\/sup>.<\/p>\n

Supporting Your Research<\/b><\/h3>\n

As spatial-omics technologies evolve, tools that streamline panel design and ensure experimental success are more important than ever. FluoroFinder supports researchers with user-friendly, cloud-based tools designed for high-parameter experiments. Our Panel Builder<\/a> helps you efficiently design and visualize complex panels for techniques like IMC\u2122, FACS, and fluorescent imaging, while our Antibody Search<\/a> platform aggregates validated antibody data from all suppliers, enabling quick comparison of clones and conjugates for multiplex studies. Whether you’re planning a spatial transcriptomics experiment or integrating protein and RNA detection using IMC and RNAscope\u2122, FluoroFinder’s tools can help you make informed decisions and accelerate discovery.<\/p>\n

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References:\u00a0<\/strong><\/h4>\n
    \n
  1. ncbi.nlm.nih.gov\/36514162\/<\/a><\/li>\n
  2. ncbi.nlm.nih.gov\/23818604\/<\/a><\/li>\n
  3. ncbi.nlm.nih.gov\/33376221\/<\/a><\/li>\n
  4. ncbi.nlm.nih.gov\/35115587\/<\/a><\/li>\n
  5. ncbi.nlm.nih.gov\/31427819\/<\/a><\/li>\n
  6. ncbi.nlm.nih.gov\/30078711\/<\/a><\/li>\n
  7. org\/content\/10.1101\/2021.06.06.447244v1<\/a><\/li>\n
  8. ncbi.nlm.nih.gov\/35143307\/<\/a><\/li>\n
  9. ncbi.nlm.nih.gov\/32015496\/<\/a><\/li>\n
  10. ncbi.nlm.nih.gov\/30479912\/<\/a><\/li>\n<\/ol>\n

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    Single-cell analysis techniques such as fluorescence-activated cell sorting (FACS) and single-cell DNA and RNA sequencing have broadened researchers\u2019 understanding of cell biology in health and disease by enabling the study of rare cell types and phenotype variations within same-cell populations. However, new methods are evolving rapidly, driven by the need for molecular profiling of individual […]<\/p>\n","protected":false},"author":8,"featured_media":12625,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"

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    The widespread adoption of fluorescence applications is a testament to the importance of fluorescent dyes in biochemical and biological research. The speed, reproducibility, and sensitivity of fluorescence-based analysis methods explain their rapid adoption in all diagnostic and research labs.\u00a0 Innovations in fluorescence technologies are still driving the development and optimization of fluorescence dyes for advanced cellular and molecular analysis. Here is a brief overview of how it all began and the pioneers that made it possible.<\/span><\/p>

    \u00a0<\/p>

    Definition of Fluorescent dye\u00a0<\/b><\/h2>

    A fluorophore is an organic molecule that emits photons upon excitation by electromagnetic radiation.<\/span> The principle behind this process is known as fluorescence. It <\/span>is based on the ability of a molecule to absorb light energy to reach an excited state. The excited molecule rapidly (1-10 ns) returns to the ground state emitting the excess energy as light at a longer wavelength. Part of the energy absorbed during excitation is dissipated by molecular quenching or intersystem crossing. Lower energy photons (longer wavelengths, lower frequency) are thus emitted. This is known as the Stokes shift and explains the differences between the excitation and the emission spectrum of a fluorophore.\u00a0<\/span><\/p>

    \u00a0<\/p>

    \"Jablonky<\/span><\/p>

    Figure 1<\/strong>. Jablonski diagram -As a fluorescent molecule absorbs energy from incident light, the energy state is excited from a stable low energy ground state (S0) to several unstable higher energy levels. Within nanoseconds, some energy is lost to non-radiative emission and the molecule adopts a lower energy state (S1). The molecule then returns to the ground state emitting light at lower energy. This shift in wavelength between absorption and emission is known as a Stokes shift.<\/span><\/h6>

    \u00a0<\/p>

    Let there be light<\/b><\/h2>

    Long before fluorescent dyes became indispensable tools in chemical biology <\/span>Bernardino de Sahag\u00fa, a <\/span>16th-century <\/span>Franciscan missionary and ethnographer, <\/span>observed that <\/span>Lignum nephriticum<\/span><\/i> (<\/span>Latin<\/span> for \"kidney wood\"), a traditional Aztec <\/span>diuretic<\/span> derived from the <\/span>wood<\/span> of two <\/span>tree<\/span> species<\/span>, (the narra, <\/span>Pterocarpus indicus<\/span><\/i>, and the Mexican kidneywood, <\/span>Eysenhardtia polystachya<\/span><\/i>) was capable of inferring fascinating opalescent hues to the water it came in contact with. This fluorescence emission was later attributed to <\/span>matlaline, the oxidation product of one of the flavonoids found in this wood [1]. <\/span>Although other observations of the phenomenon were reported, the term \u201cfluorescence\u201d was coined only 300 years later by George Gabriel Stokes after observing the emission of light when a solution of quinine in ethanol was irradiated with UV radiation isolated with a prism [2]. <\/span>Less than 20 years later the first fluorescent molecule was synthesized by <\/span>Adolf von Baeye. By heating <\/span>phthalic anhydride and resorcinol over a zinc catalyst he obtained a deep red powder that emitted an intense yellow-green fluorescence in the presence of alkaline solutions<\/span>. Baeyer named this compound \"resorcinphthalein\" [3]. <\/span>Today it is known as <\/span>fluorescein <\/b>(figure 2) and serves as the scaffold for the <\/span>most widely used class of fluorophores: the xanthene-based fluorescent dyes.<\/span><\/p>

    \u00a0<\/p>

    \"Fluorescein_2\"<\/a><\/p>

    Figure 2: <\/strong>Chemical structure of fluorescein.<\/h6>

    \u00a0<\/p>

    Rhodamines<\/b><\/b><\/h2>

    In\u00a0 1887, the industrial chemist Ceresole [3] obtained a new class of highly fluorescent dyes with red-shifted spectra. He named them Rodhamines, from the greek rodhon, rose - the color of the novel compounds. One interesting property of rhodamines is the effect of the polarity of the solvent on their color and fluorescence emission. When dissolved in non-polar solvents the dye is in a colorless closed lactone form while the protonated open form is colored and fluorescent. This feature and the ease of synthesis make rodhamines ideal building blocks for the construction of photoactivable dyes. <\/span>Rhodamine<\/b> dyes provided greater photostability, pH insensitivity, and longer emission wavelengths relative to fluorescein. Popular rhodamine dyes include Oregon Green, Texas red, and several Alexa Fluor\u00ae dyes.<\/span><\/p>

    \"Condensation<\/p>

    \u00a0<\/p>

    Figure 3<\/b>: <\/span>Condensation reaction between 3-aminophenols and phthalic anhydride produced a new class of highly fluorescent dyes named tetramethylrhodamine.\u00a0<\/span><\/h6>

    \u00a0<\/p>

    Cy (Cyanine) dyes<\/b><\/h2>

    In the 1970s, the chemist and amateur photographer Alan Waggoner began his quest to synthesize photostable, water-soluble, and non-cytotoxic dyes. Inspired by the cyanine family of dyes used at the time to produce colors on photographic films, and despite being color-blinded, he began systematically modifying the structure of cyanine dyes to make them successful for use in living cells.\u00a0<\/span>His work produced the cyanine (Cy) family of dyes, which includes Cy3 and Cy5, some of the most commonly used fluorochromes in biomedical research. Cyanines increased brightness and photostability made them a great alternative to conventional dyes such as <\/span>fluorescein<\/span> and rhodamine<\/span>. For example, Cy5 became a popular replacement for far-red fluorescent dyes because of its high extinction coefficient and its fluorophore emission maximum in the red region where many CCD (Charge Coupled Device) detectors<\/span><\/span> have maximum sensitivity and biological objects give low background interference. [6]<\/a><\/span><\/p>

    \u00a0<\/p>

    Bioconjugation<\/b><\/h2>

    One of the main applications of fluorescent dyes is for labeling biomolecules. The attachment of fluorescent probes to biomolecules is the basis of any experiment using fluorescence detection. The most widely used fluorescent bioconjugates are labeled-antibodies. Used in numerous applications including flow cytometry, immunofluorescence, microarrays, fluorescence ELISA,\u00a0 western blotting, and super-resolution microscopy, conjugated antibodies are indispensable tools in any research lab.\u00a0<\/span><\/p>

    \u00a0<\/p>

    Conclusions<\/b><\/h2>

    The increasing adoption of fluorescence-based multiplex analysis methods and the emergence of super-resolution imaging techniques continue to drive the development of new dyes with improved photochemical properties. <\/span>Increased brightness and photostability, higher molar extinction coefficient and quantum yield, long fluorescence lifetime,\u00a0 increased water solubility, and narrow emission profiles are highly sought characteristics in the newest generation of dyes.\u00a0<\/span>Of particular interest is the expansion of available dyes with emission in the near-infrared range (NIR) as they provide not only a\u00a0 way to build increasingly larger flow cytometry panels but also offer strong tissue penetration and low phototoxicity for fluorescent imaging of complex biological samples [7].\u00a0<\/span><\/p>

    \"fluorescent<\/p>

    Figure 4:<\/strong> Number of <\/span>yearly publications on fluorescent dyes.<\/h6>

    \u00a0<\/p>

    FluoroFinder\u2019s platform<\/a> provides access to the tools and resources needed to design fluorescence experiments and to choose the best <\/span>dyes<\/span><\/a> and <\/span>conjugated antibodies<\/span><\/a> for fluorescence analysis and detection. <\/span>For example,<\/span> \u00a0FluoroFinder\u2019s <\/span>\u00a0<\/b>spectra viewer<\/b><\/a> can be used to:<\/span><\/p>