By David Wold
Spatial biology, or spatial omics, allows the visualization and study of the interactions of individual molecules, tissues, and cells in three dimensions, over time.1-4 This can help us to better understand biological processes, development, and disease mechanisms in their natural context, and identify biomarkers and potential therapeutic targets. The first article of our series featured an introduction to spatial omics, the second focused on spatial transcriptomics, and in this final article, we explore how Tecan’s Cavro liquid handling solutions can be used in spatial omics platform development and automation.
MERSCOPE image of human kidney tissue, revealing the striking arrangements of tubular structures, with the spatially resolved expression of 11 genes (image copyright Vizgen Inc.)
Liquid handling in spatial transcriptomics workflows
In our second article, we looked specifically at the standardization of spatial transcriptomics workflows: multiplexed error robust FISH (MERFISH*), sequential FISH (seqFISH), and seqFISH+. All these techniques utilize combinatorial labeling schemes to assign each transcript to a barcode, which consists of a subset of hybridization probes that are detected in repeated cycles of imaging and release.2,5,6
With the increased throughput enabled by these emerging spatial transcriptomics technologies, looking at the expression of 100s of genes over many cycles of hybridization, there is a huge potential for liquid-handling-induced errors. The precision of liquid handling directly impacts data quality, reproducibility, and the ability to detect rare transcripts across tissue sections.
As an example, Figure 1 highlights the many liquid handling steps in the basic MERFISH workflow. These stretch from sample collection and preparation to the multiple cycles of hybridization before data collection and analysis. Reliable automation of these liquid handling steps is thus arguably the most important criterion for achieving successful outcomes in MERFISH-based transcriptomics studies.
Figure 1: Overview of the liquid-handling steps required for 16 successive MERFISH hybridizations (Moffitt and Zhuang, 2016).7
As the MERFISH workflow is so complex, minor variations in sample type, sample handling, probe preparation, staining protocols, and instrumentation can potentially introduce unwanted differences in results, particularly regarding coverage, background noise, and sensitivity.
Despite this complexity, MERFISH and its variants provides us with a surprising number of intrinsic advantages that contribute to its potential for standardization. These include but are not limited to:
High multiplexing capacity: MERFISH can detect thousands of RNA species simultaneously in individual cells, allowing comprehensive transcriptome profiling.
Probe design and encoding strategy: MERFISH uses a combinatorial labeling approach, where each RNA species is encoded by a unique combination of fluorescent probes. This encoding strategy allows for error correction, improving the accuracy of RNA detection and quantification.
Single-molecule resolution: the technique enables the visualization and quantification of individual RNA molecules while providing precise spatial information.
Scalability: MERFISH can be applied to large tissue sections, making it suitable for analyzing complex organs and even whole organisms.
Compatibility with tissue preservation methods: MERFISH works well with fixed tissues, allowing for retrospective analysis of archived samples.
Using the Cavro XCalibur Pump for MERFISH: a case study
With all its advantages, MERFISH and its derivatives have the potential to become go-to techniques for spatial transcriptomics. However, for this to happen, the liquid handling protocols must be completely reliable. Liquid handling solutions that might be considered include the Tecan Cavro range of pumps.
To this end, the Cavro XCalibur (XC) Pump has already been successfully integrated into Vizgen’s MERSCOPE® Platform, a high multiplexed, high-resolution in situ MERFISH-based solution that combines single-cell and spatial genomics analysis. The MERFISH technology allows researchers to look at a large number of gene targets – currently up to 1000 – on the MERSCOPE Platform, with high specificity and sensitivity. Cleverly designed, error-robust barcodes make it possible to efficiently distinguish between targets and correct readout errors, if detected.
The workflow resembles single-molecule FISH. Tissue samples are sectioned and mounted on a proprietary MERSCOPE Slide – a slide designed for optimal optical clarity. The workflow operations vary slightly based on the sample type – fresh, frozen, cell culture, or FFPE. However, the major steps are consistent: sectioning, hybridization, gel embedding and clearing, followed by MERFISH imaging. The clearing process is designed to digest the tissue components, which minimizes unwanted background fluorescence while preserving the RNA. This technique is specific to MERFISH and MERSCOPE. By reducing background fluorescence, these methods enhance the ability to accurately detect the target RNA transcripts.
After sample preparation, the slide is placed into the flow chamber of the MERSCOPE instrument, and the entire imaging and analysis process is automated, including multiple rounds of hybridization with reporter probes. A low magnification (10x) overview of the entire tissue section is presented to the user, allowing them to select specific areas or the whole tissue for imaging. The data then undergo automatic cell segmentation and transcript analysis. Throughput is maximized by loading the next sample to be imaged onto the platform while analysis of the previous run completes.
The success of any automated system depends on reliable, accurate and precise pumping technology. To this end, Vizgen chose the Cavro XC pump for the MERSCOPE fluidics system. The pump moves liquid from the reagent cartridge into the fluid chamber, and subsequently to waste, and is typically used to move volumes from tens of microliters up to a few milliliters at each stage, with imaging performed between fluidic steps.
MERSCOPE enables researchers to understand which gene expression networks are active and inactive, and which genes are upregulated and downregulated, in different cellular and disease states. The MERSCOPE Ultra™ Spatial Imaging Platform can map transcripts across 3 cm2 of tissue on a single slide, offering a tissue-wide view of up to 1000 custom genes at single-cell resolution.
The value and flexibility of the underlying MERFISH technique has been demonstrated in a variety of applications, including characterization of the molecular homogeneity of the mouse brain and illumination of the tumor microenvironment, while other researchers in the field have generated publications across disciplines such as neurology, oncology, immunology, and infectious diseases.8,9 More information can be found on the Vizgen website.
Cavro pumps: multiple roles in spatial omics automation
With its high precision and accuracy, small footprint, and plug-and-play nature, the XC pump plays an obvious key role at the heart of MERFISH and related techniques, with their multiple cycles of hybridization, imaging, washing, and re-probing.
Other features that make Cavro pumps ideal for spatial omics workflow automation include their precise reagent delivery, from nanoliters to milliliters, and their programmable flow rates, so that reagent delivery is consistent across tissue sections while at the same time protecting sample integrity. Precisely controlled dispensing speeds are also important when dealing with reagents of varying viscosities and sensitive solutions, such as antibodies.
The integration capabilities of Cavro pumps allow the simple automation of otherwise complex, overlapping liquid handling sequences, enabling parallel sample processing. The pumps are also chemically compatible with both aqueous and non-aqueous solutions, and their temperature control features support a wide range of applications, from simple buffer exchanges to sensitive enzymatic reactions.
Cavro pumps have an intrinsically low dead-volume design, which minimizes the waste of expensive reagents while ensuring efficient sample use and reducing cross-contamination. The pumps maintain their robust performance and high precision over extended operation periods with minimal maintenance requirements, making them an exceptionally dependable choice for automating demandingly repetitive spatial omics protocols. And finally, built-in sensors enable continuous flow monitoring and error detection, supporting GMP compliance in regulated environments and paving the way for their potential future use in the clinic.
Expanding your applications into spatial omics: what next?
In summary, the use of the Cavro pump range in the automation of spatial transcriptomics workflows ensures:
- Precise volume control down to nanoliter scales
- Consistent reagent dispensing across tissue sections
- Reduced operator-dependent variation
- Standardized hybridization and washing protocols
However, if you are not using or adapting an existing platform, you may need more than an industry-leading range of pumps when developing your new automated spatial omics application. Tecan also provides the Cavro OEM liquid handling range to fast-track such instrument development, which includes robots, pipettors, and other components.
Tecan Cavro liquid handling robots: the foundation for your OEM instrument. Various standard and custom configurations are available, to provide automated liquid handling solutions for almost any OEM application, using fixed or disposable tips. With over 40 years of experience, Tecan's quality workmanship, understanding of market needs, and expertise in OEM component design help to ensure Cavro robots suit virtually any application.
Cavro Omni Flex: a complete solution for developing liquid handling platforms. Combining the proven liquid handling performance of the Cavro Omni Robot with a purpose-built chassis and worktable, it provides a modular, convenient solution for automating pipetting and sample handling activities.
Quality standards with comprehensive compliance. The need for extensive component testing can hold up the release of new products, particularly in the IVD market. The Cavro Omni Flex is designed and manufactured to the highest quality standards, including ISO13485, ISO9001 and FDA 21 CFR Part 820 requirements. In addition, Cavro components follow national and regional compliance – such as Europe’s RoHS Directive, REACH and Conflict Minerals requirements – with documentation and regulatory certificates available to support compliance activities for your new spatial omics application.
Ready to start doing spatial omics? Talk to our Cavro OEM team.
*The MERFISH technique was pioneered in the laboratory of Xiaowei Zhuang, Harvard University, Cambridge, Massachusetts.7 The MERFISH patents are owned by The President and Fellows of Harvard College. Patent No. 11,959,075, relating to robust multiplex imaging of RNA and its spatial organization in a sample, is exclusively licensed to Vizgen.10,11
References
1. Alexandrov, T., Saez-Rodriguez, J., & Saka, S. K. (2023). Enablers and challenges of spatial omics, a melting pot of technologies. Molecular systems biology, 19(11), e10571.
Pubmed: https://pubmed.ncbi.nlm.nih.gov/37842805/
DOI: https://doi.org/10.15252/msb.202110571
2. Bressan, D., Battistoni, G., & Hannon, G. J. (2023). The dawn of spatial omics. Science (New York, N.Y.), 381(6657), eabq4964.
Pubmed: https://pubmed.ncbi.nlm.nih.gov/37535749/
DOI: https://doi.org/10.1126/science.abq4964
3. https://pubmed.ncbi.nlm.nih.gov/?term=%22spatial+omics%22&filter=years.2020-2024&sort=date (Accessed 11 November 2024)
4. Marx, V. Method of the Year: spatially resolved transcriptomics. Nat Methods 18, 9–14 (2021).
DOI : https://doi.org/10.1038/s41592-020-01033-y
5. Eng, C. L., Lawson, M., Zhu, Q., Dries, R., Koulena, N., Takei, Y., Yun, J., Cronin, C., Karp, C., Yuan, G. C., & Cai, L. (2019). Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature, 568(7751), 235–239.
Pubmed: https://pubmed.ncbi.nlm.nih.gov/30911168/
DOI: https://doi.org/10.1038/s41586-019-1049-y
6. Lu, T., Ang, C. E., & Zhuang, X. (2022). Spatially resolved epigenomic profiling of single cells in complex tissues. Cell, 185(23), 4448–4464.e17.
Pubmed : https://pubmed.ncbi.nlm.nih.gov/36272405/
DOI : https://doi.org/10.1016/j.cell.2022.09.035
7. Moffitt, J. R., & Zhuang, X. (2016). RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH). Methods in enzymology, 572, 1–49.
PubMed : https://pubmed.ncbi.nlm.nih.gov/27241748/
DOI : https://doi.org/10.1016/bs.mie.2016.03.020
8. Vizgen. Application note: Distinguish the molecular and cellular heterogeneity of the mouse brain with the MERSCOPE PanNeuro Cell Type Predesigned Gene Panel.
https://info.vizgen.com/resource-download-panneuro-panel-app-note
(Accessed 11 November 2024.)
9. Vizgen. Application note: Illuminate the tumor microenvironment and understand oncological heterogeneity with the MERSCOPE PanCancer Pathways Panel for Human Tissue.
https://vizgen.com/resources/illuminate-the-tumor-microenvironment-and-understand-oncological-heterogeneity-with-themerscope-pancancer-pathways-panel-for-human-tissue-application-note (Accessed 11 November 2024.)
10. https://patents.google.com/patent/US11959075B2/en?oq=US11959075
(Accessed 11 November 2024.)
11. https://vizgen.com/vizgen-issued-a-new-us-patent-for-its-merfish-technology/
(Accessed 11 November 2024.)
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About the author
David Wold
David Wold is the Customer Operations Manager for the Partnering Business in the Components Marketing Team. In this role, he oversees a team responsible for the support and satisfaction of Tecan’s top Components customers. Since 2005, David has helped Tecan customers meet their liquid handling challenges across a broad range of technologies and applications. He brings understanding and expertise to help customer choose components to meet their needs, provide maximum reliability and outstanding performance.