An Overview of Multiplex Slide Imaging Approaches

At Visikol, our core objective is to help service Clients and product Customers to transform their tissues into quantitative data sets that can be mined for actionable insights in order to answer a specific research question. To meet this objective, we employ a wide range of tissue imaging approaches from plate reading to light sheet microscopy and leverage a diverse suite of labeling and imaging analysis tools.

While we will often work with Clients on H&E and IHC projects, much of our work is focused on multiple channel fluorescent imaging either in a traditional 2D format (i.e. slide sectioning) or in a 3D format using tissue clearing and 3D microscopy (e.g. light sheet, confocal). While H&E and IHC are gold-standards for histopathology, they limit the dimensionality of the data that can be collected from slides as separating multiple chromogenic stains from brightfield imaging can be challenging and thus imaging a panel of immuno-oncology markers at the same time from one slide is not feasible unless serial sections are used and aligned which can be problematic from a co-registration and cost perspective.

Approaches for Multiplex Slide Imaging – Fluorescence vs Mass Cytometry Imaging


There are currently two fundamental approaches for multiplex slide imaging: fluorescence and imaging mass cytometry (i.e. CyTOF). Fluorescence involves the use of multiple fluorescent markers which due to their excitation and emission wavelengths (i.e. Stokes Shift) allow for multiple markers to be used simultaneously through using different excitation and emission wavelength filters. While relatively inexpensive and easy-to-use with 3-5 fluorescent channels, fluorescent approaches are limited in that once fluorescent spectra overlap, there can be considerable noise which makes isolating channels from one another challenging without the use of sophisticated deconvolution techniques. Below, we describe several different approaches to achieve greater than 5-plex tissue labeling and imaging.

The other approach for slide multiplexing is to use what is referred to as mass cytometry. In this approach, tissues are labeled with antibodies that have heavy metal isotope conjugated to them which do not naturally occur within animal tissues. With the Fluidigm Hyperion Imaging System, tissues are ablated using a 1 um2 plasma arc and the resulting metal isotopes are run through a mass spec from each individual 1 um2 pixel. The mass spec data from each pixel can then be used to create imaging data as the mass spec readouts are correlated with the quantity of a specific metal isotope (i.e. biomarker) within a tissue pixel.

Fluorescent Based Multiplex Tissue Imaging Approaches

1. Tissue-Based Cyclic Immunofluorescence (t-CyCIF) Method:

This technique involves a low plex staining and imaging of tissue sample with fluorescently conjugated primary antibodies. This is followed by bleaching the fluorescent signal and labelling the sample with the next set of fluorescently conjugated antibodies. This cycle can be repeated several time and studies show that the process has been successfully repeated at least 15 times without any damage to tissue morphology or loss of antigenicity.

2. Co-Detection by Indexing or Fluorescent Immunohisto-PCR:

In this approach, tissue samples are incubated with antibodies conjugated to DNA containing a unique 5’ overhang sequence. In a PCR like approach, cells are exposed to a nucleotide mix that contains one of two non-fluorescent “index” nucleotides (A and G) and two fluorescent labeling nucleotides (U and C). The index nucleotides fill in the first index position across all antibodies bound to the cells. The technology is designed in such a way that only the first two antibodies are capable of being labeled with one of the two fluorescent nucleotides only if the index nucleotide was previously incorporated. Those two antibodies are then imaged by standard fluorescence microscopy and the fluorophores are cleaved by TCEP mediated stripping, washed away, and the sample is ready for the next cycle where a different indexing nucleotide is used.

3. Fluorescent Tyramide Mediated Amplification:

This technique involves conventional IHC-HRP based protocol, but instead of using DAB as the substrate, Tyramide labelled fluorophores are used as a substrate. Once the HRP reaction is initiated in the presence of hydrogen peroxide, the tyramide labelled fluorophore gets covalently linked to tyrosine residues in the vicinity of the primary-secondary antibody complex. Using this approach several groups have sequentially labelled samples with up to 8 biomarkers. This approach however requires the use of spectrally distinct fluorophores and special spectral scanners.

4. Cyclic Removal of Antibodies

This approach has been pioneered by Visikol and leverages an antibody stripping reagent called EasyPlex that is capable of removing antibodies from slides. Tissues are labeled with 3-5 antibodies with DAPI and then imaged followed by antibody removal. The tissue can then be relabeled and imaged multiple times without tissue degradation which allows for 10+ label imaging. After each panel is imaged, the resulting datasets are co-registered to one another using the DAPI nuclear channel. This approach does not require special antibodies and leverage existing imaging equipment which makes it inexpensive and east to adopt.

Advantages and Disadvantages of Multiplex Techniques


The primary disadvantage of fluorescent multiplex techniques is that cross-talk between channels can impart noise into a dataset and thus reduce the overall quality of the data generated. While labels can be stripped for increased dimensionality, this process requires robust validation to ensure that the washing away of labeling rounds does not disrupt epitopes or overall tissue morphology. While thee are several companies that describe proprietary techniques with 20+ label capabilities, the limitation with many of these techniques is cost and the need to validate for every single tissue, fixation approach and new panel of labels.

With mass cytometry, 40+ multiplexing is feasible without any cross-talk between markers due to the nature of mass spec detection (i.e. time of flight) compared to fluorescent detection. However, the downside of this approach is that it only provides 1 um image pixels (i.e. equivalent to 10X imaging) and takes 2 hours per 1 mm2 area on a slide. Conversely, whole slide fluorescent 40X imaging with 4 channels can image a whole slide in 60-90 minutes.

Which Approach for Multiplexing Should You Use?

Using standard off-the-shelf antibodies and imaging instruments, most researchers can achieve 3-5 plex imaging in their lab using fluorescent detection. The challenge starts when a researcher tries to go above five as either it requires the use of a technique like imaging mass cytometry, DIY approaches such urea-based stripping or proprietary techniques. The challenge with imaging mass cytometry is that is requires a highly expensive instrument as well as metal-conjugated antibodies and a lot of consumables (e.g. argon). This makes the cost per sample of imaging mass cytometry relatively high wherein the cost of materials alone can be as much as $800 per 1 mm2  image with a dozen markers. Imaging mass cytometry is therefore recommended for applications where a tissue is precious and you NEED to get 15 plus markers from a single slide with minimum background noise as some of your proteins might have low expression that would blend into the background with fluorescent imaging.

Over the last few years with the explosion of immuno-oncology therapeutic research and the desire to study multiple immune cell sub-types from a single slide, there have been a wide range of multiplexing protocols described in the literature for fluorescent imaging. However, the exact approach that you take to multiplexing in your lab depends on a balance between cost, throughput and validation. We find that a lot of times while an imaging approach is technically feasible such as imaging a whole mouse brain at 40X using light sheet microscopy, there are typically better ways to address the same research question while minimizing complexity and cost. For example, using two serial sections with five labels each will allow you to achieve ten-plex imaging appropriate for most research questions without requiring expensive and troublesome commercial approaches.

If you are interested in learning more about multiplexing or applying it to your next research project, reach out to us today to discuss our multiplex and advanced imaging capabilities.

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