CiteAb introductory guides: Multiplexed antibody based imaging techniques
In this blog:
- We provide an overview of multiplexed antibody based imaging
- We give some examples of different antibody tags used in the technique
- We discuss the drawbacks and benefits of the method
Multiplexed antibody based imaging is an incredibly powerful tool which provides a spatial understanding of cellular structure, cellular communication and composition in situ.
Inspired by an insightful table we came across in Nature Methods and Protocols, in our blog today we give an overview of multiplexed antibody based imaging techniques, to give you a starting point when orienting yourself in this field. 
What is multiplexed antibody based imaging?
Traditional microscopy methods such as IHC and ICC typically allow you to image under five targets. However, this will not give the clearest picture of the cellular world. The more targets available, the more complexity we are able to understand.
Multiplexed antibody based imaging methods use different antibody tags, staining and detection methods to enable more targets to be detected in one single sample.
Our introductory overview to multiplexing using antibody based imaging splits methods by the type of label used on antibodies: fluorophore-labelled, DNA-labelled and metal-labelled. We outline the concept of each method and delve into some examples.
Fluorescence-labelled antibody based multiplex imaging
Using fluorescent-conjugated antibodies for imaging is a widely established methodology in research.
Spectral overlap of fluorophores means that currently only four to seven epitopes can be detected at once with traditional imaging techniques. To overcome this limitation, multiplex imaging using fluorophore conjugated antibodies often makes use of cyclic staining.
The tissue of interest is stained, imaged, then the antibody-conjugates removed and the next cycle of staining begins. To achieve this cyclic type of staining, different methods can be utilised including dye inactivation or antibody removal. This means many epitopes can be detected; as many as 100 have been reported in one method.
IBEX: iterative bleaching extends multiplicity
The IBEX method can detect over 65 targets by using a cyclic approach to staining, involving iterative rounds followed by chemical bleaching to inactivate the dye. Staining can take around 30-45 minutes, and bleaching around 15. 
A benefit of this method is its versatility. Off the shelf antibodies can be used, as well as conventional microscopes rather than specialised instruments. IBEX can also detect fluorescent proteins expressed by animal models through genetic engineering.
Opal IHC has the ability to detect fewer targets: around 6-8. This approach uses complete antibody removal, instead of dye inactivation, to achieve multiplexing. Antibody conjugates used can be fluorescent or chromogenic. 
Although fewer targets can be detected with this method, the antibody staining approach makes use of tyramide signal amplification. This can help to improve signal to noise ratios and enable the detection of isotopes with particularly low abundance. It is worth noting that these tyramide linked fluorophores can be challenging to remove from tissues, although recent advances may reduce this issue.
Other techniques include:
- Vibrational (over 20 targets)
- MELK (100 targets)
- tCyCIF (60 targets)
- And many more
Find out more
General overview: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9264278/
IBEX overview: https://www.pnas.org/doi/10.1073/pnas.2018488117
Detail on Opal IHC: https://febs.onlinelibrary.wiley.com/doi/full/10.1002/1878-0261.12764
Antibody-oligonucleotide conjugates for multiplex imaging
In this methodology, antibodies conjugated to a short oligonucleotide are used for multiplex imaging. The concept is very similar to multiplexing using fluorescent labelled antibodies, but instead of completely removing the antibody in each cycle of staining, oligos are bound and unbound from antibodies to enable more targets to be detected and all-in-one staining to be carried out.
CODEX (co-detection by indexing)
CODEX uses a DNA barcode label in order to detect up to 60 targets, with fluorescent dyes targeting specific oligo sequences for visualisation. After a cycle of labelling, imaging and reporter removal (making use of an automated fluidics system) a full picture of the sample is built up. 
Immuno-SABER (immunostaining with signal amplification by exchange reaction)
The immuno-SABER method involves an amplification method based on primer exchange reactions to increase sensitivity. Multiplexing is achieved by DNA exchange imaging (staining with DNA barcoded antibodies followed by buffer exchange of DNA imager strands with a fluorophore attached) and the detection method is cyclic. Using this method will allow you to detect over ten targets. 
Other techniques include:
Find out more
Details on CODEX: https://protocolsmethods.springernature.com/posts/codex-multiplexed-imaging-entering-the-age-of-technicolor-histology
Details on Immuno-SABER: https://pubmed.ncbi.nlm.nih.gov/31427819/
Heavy metal labelled antibody based imaging techniques
An alternative to approaches requiring fluorescence is the use of heavy metals. Using this technique overcomes many of the limitations associated with fluorescence, such as background signal and fluorescent dye spillover. On top of this, the approach to staining with this method is all-in-one.
Multiplex imaging using heavy metal conjugated antibodies works by using an instrument such as a laser to generate a stream of particles from the samples tagged with heavy metals. The second instrument (such as a Cytof instrument, which separates metal ions based on mass) then understands the masses of the isotopes on the particles in order to build the image of the sample.
Using heavy metal conjugated antibodies is not without its limitations. At this time, instruments used in this approach are more specialised, which can limit its applicability across many laboratories. Antibody labels cannot be amplified by this method, image acquisition may be slow, and it can become limited by access to isotopically pure lanthanide metals. 
MIBI-TOF (Multiplexed Ion Beam Imaging)
MIBI-TOF can detect over 40 targets in an all-in-one antibody staining approach, however it is time consuming, costly and less sensitive than some other techniques. Tissues are FFPE stained or frozen. Upon applying an ion beam to the tissue, secondary metal isotope ions are released and can be detected. 
IMC (Imaging Mass Cytometry)
IMC makes use of CyTOF technology (cytometry by time of flight) which combines both flow cytometry and mass spectrometry. It utilises high resolution scanning laser ablation to generate the particles for detection via CyTOF, and can detect over 40 targets. 
Find out more:
MIBI details: https://www.ionpath.com/mibi-technology/
IMC details: https://bmcophthalmol.biomedcentral.com/articles/10.1186/s12886-021-02099-8
Compare antibodies in our reagent search engine
You can use the CiteAb antibody search engine to find particular antibodies you want to use in these methods, or for other experiments in your lab. The search will return results from many different suppliers, ranked by the number of citations in the scientific literature.
You can filter by antibody-conjugate, such as fluorescent-conjugated or oligo-conjugated, and often by specific application. We have many filters available to further refine searches, such as suppliers, validations, host and more.
Try it out here:
You may be wondering why we need these technologies, why not just perform imaging on six sections of the cell? The answer is that this can be technically impossible given cell size, and in clinical settings tissue conservation may be critical so that other tests can be carried out. Multiplexing overcomes these challenges.
That is not to say that the technique doesn’t come with its own challenges. Potential issues can include: lengthy cycles (up to hours can be consumed by a single cycle), signal decay, and quenching being incomplete which can compromise results.
The importance of antibody validation is, of course, also prominent here. When using multiple antibodies, it is important they are specific to their target, and for the application.
In spite of this, multiplex antibody based imaging has proved to be very important for research into tissue biology, oncology, drug development and biomarker discovery, to name just a few areas. The likely future impact of this technique, still relatively new and novel, is significant. We are excited to see what lies ahead.
Stay tuned for more introductory guides in the future on interesting and new areas in the life sciences!
- Skye and the CiteAb team
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- Radtke, A.J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C.J., Gola, A., Thakur, N., Shih, R., Yao, L., Yaniv, Z.R., Beuschel, R.T., Kabat, J., Croteau, J., Davis, J., Hernandez, J.M. and Germain, R.N. (2020). IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National Academy of Sciences, 117(52), pp.33455–33465. doi:https://doi.org/10.1073/pnas.2018488117.
- Viratham Pulsawatdi, A., Craig, S.G., Bingham, V., McCombe, K., Humphries, M.P., Senevirathne, S., Richman, S.D., Quirke, P., Campo, L., Domingo, E., Maughan, T.S., James, J.A. and Salto-Tellez, M. (2020). A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment. Molecular Oncology, [online] 14(10), pp.2384–2402. doi:https://doi.org/10.1002/1878-0261.12764.
- Community, S.N.P. and M. (2021). CODEX Multiplexed Imaging – Entering the Age of Technicolor Histology. [online] Springer Nature Protocols and Methods Community. Available at: https://protocolsmethods.springernature.com/posts/codex-multiplexed-imaging-entering-the-age-of-technicolor-histology#:~:text=CODEX%20is%20a%20high%2Dparameter [Accessed 16 Feb. 2023].
- Saka, S.K., Wang, Y., Kishi, J.Y., Zhu, A., Zeng, Y., Xie, W., Kirli, K., Yapp, C., Cicconet, M., Beliveau, B.J., Lapan, S.W., Yin, S., Lin, M., Boyden, E.S., Kaeser, P.S., Pihan, G., Church, G.M. and Yin, P. (2019). Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues. Nature Biotechnology, [online] 37(9), pp.1080–1090. doi:https://doi.org/10.1038/s41587-019-0207-y.
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- Schlecht, A., Boneva, S., Salie, H., Killmer, S., Wolf, J., Hajdu, R.I., Auw-Haedrich, C., Agostini, H., Reinhard, T., Schlunck, G., Bengsch, B. and Lange, C.A. (2021). Imaging mass cytometry for high-dimensional tissue profiling in the eye. BMC Ophthalmology, 21(1). doi:https://doi.org/10.1186/s12886-021-02099-8.